Publications
2024
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(2024) Structure (London, England : 1993). 32, 10, p. 1563-1580 Abstract[All authors]
Instruct-ERIC, "the European Research Infrastructure Consortium for Structural biology research," is a pan-European distributed research infrastructure making high-end technologies and methods in structural biology available to users. Here, we describe the current state-of-the-art of integrated structural biology and discuss potential future scientific developments as an impulse for the scientific community, many of which are located in Europe and are associated with Instruct. We reflect on where to focus scientific and technological initiatives within the distributed Instruct research infrastructure. This review does not intend to make recommendations on funding requirements or initiatives directly, neither at the national nor the European level. However, it addresses future challenges and opportunities for the field, and foresees the need for a stronger coordination within the European and international research field of integrated structural biology to be able to respond timely to thematic topics that are often prioritized by calls for funding addressing societal needs.
2023
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(2023) Acta Crystallographica Section D: Structural Biology. 79, Pt 11, p. 992-1009 Abstract[All authors]
A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38 Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P-O bonds with the exposed β-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand-protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase-organophosphate structures.
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(2023) FEBS Journal. 290, 13, p. 3383-3399 Abstract[All authors]
Acid-β-glucosidase (GCase, EC 3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme®) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step towards developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine-learning based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.
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(2023) Proteins: Structure, Function and Bioinformatics. 91, 8, p. 1097-1115 Abstract
\u201cNewly Born\u201d proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by the expression of novel open reading frames, and appear throughout evolution. We were curious if three recently developed programs for predicting protein structures, namely, AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such \u201cNewly Born\u201d proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called \u201cNever Born\u201d proteins. The programs were used to compare the structures of two sets of \u201cNever Born\u201d proteins that had been expressedGroup 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, although the models generated were scored as being of low quality, they nevertheless revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms, in agreement with the experimental data, whereas the members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins, again consistent with the experimental data. These predicted differences were shown to be statistically significant by comparing their accessible surface areas. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Surprisingly, only for the protein which did not have a novel fold, and was taxonomically restricted, rather than being a true orphan, did all three algorithms predict very similar, high-quality structures, closely resembling the crystal structure. Finally, they were used to predict the structures of seven orphan proteins with well-identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with only two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high-quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member, and to the fact that the Dali server revealed several nonrelated proteins with similar folds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Proteins:3.
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(2023) Nucleic Acids Research. 51, 2, p. 806-830 Abstract[All authors]
Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.
2021
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(2021) Biochemistry and Molecular Biology Education. 49, 5, p. 707-719 Abstract[All authors]
Proteopedia (proteopedia.org) is an open resource to explore the structurefunction relationship of proteins and other biomolecules. This guide provides practical advice on how to incorporate Proteopedia into teaching the structure and function of proteins and other biomolecules. For 11 activities, we discuss desired outcomes, setting expectations, preparing students for the tasks, using resources within Proteopedia, and evaluating student work. We point out features of Proteopedia that make it especially suitable for teaching and give examples of how to avoid common pitfalls.
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(2021) Protein Science. Abstract
Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca+2, Mg+2 and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one highaffinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several waters molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of rmsd values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins.
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(2021) Acta Crystallographica Section D: Structural Biology. 77, 2, p. 151-163 Abstract[All authors]
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank.
2020
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(2020) Neuropharmacology. 179, 108265. Abstract
Computational approaches have proved valuable in elucidating structure/function relationships in the cholinesterases in the context of their unusual three-dimensional structure. In this review we survey several recent studies that have enhanced our understanding of how these enzymes function, and have utilized computational approaches both to modulate their activity and to improve the design of lead compounds for their inhibition. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Neuropharmacology:2
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(2020) F1000Research. 9, 278. Abstract[All authors]
Structural bioinformatics provides the scientific methods and tools to analyse, archive, validate, and present the biomolecular structure data generated by the structural biology community. It also provides an important link with the genomics community, as structural bioinformaticians also use the extensive sequence data to predict protein structures and their functional sites. A very broad and active community of structural bioinformaticians exists across Europe, and 3D-Bioinfo will establish formal platforms to address their needs and better integrate their activities and initiatives. Our mission will be to strengthen the ties with the structural biology research communities in Europe covering life sciences, as well as chemistry and physics and to bridge the gap between these researchers in order to fully realize the potential of structural bioinformatics. Our Community will also undertake dedicated educational, training and outreach efforts to facilitate this, bringing new insights and thus facilitating the development of much needed innovative applications e.g. for human health, drug and protein design. Our combined efforts will be of critical importance to keep the European research efforts competitive in this respect. Here we highlight the major European contributions to the field of structural bioinformatics, the most pressing challenges remaining and how Europe-wide interactions, enabled by ELIXIR and its platforms, will help in addressing these challenges and in coordinating structural bioinformatics resources across Europe. In particular, we present recent activities and future plans to consolidate an ELIXIR 3D-Bioinfo Community in structural bioinformatics and propose means to develop better links across the community. These include building new consortia, organising workshops to establish data standards and seeking community agreement on benchmark data sets and strategies. We also highlight existing and planned collaborations with other ELIXIR Communities and other European infrastructures, such as the structural biology community supported by Instruct-ERIC, with whom we have synergies and overlapping common interests.
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(2020) Molecular Biology and Evolution. 37, 4, p. 1133-1147 Abstract[All authors]
Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the refunctionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
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(2020) Chemico-Biological Interactions. 319, 109007. Abstract
Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.
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(2020) International Journal of Molecular Sciences. 21, 5, 1683. Abstract
Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M-1 s-1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.
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(2020) Molecules. 25, 5, 1198. Abstract
Over recent decades, crystallographic software for data processing and structure refinement has improved dramatically, resulting in more accurate and detailed crystal structures. It is, therefore, sometimes valuable to have a second look at "old" diffraction data, especially when earlier interpretation of the electron density maps was rather difficult. Here, we present updated crystal structures of Drosophila melanogaster acetylcholinesterase (DmAChE) originally published in [Harel et al., Prot Sci (2000) 9:1063-1072], which reveal features previously unnoticed. Thus, previously unmodeled density in the native active site can be interpreted as stable acetylation of the catalytic serine. Similarly, a strong density in the DmAChE/ZA complex originally attributed to a sulfate ion is better interpreted as a small molecule that is covalently bound. This small molecule can be modeled as either a propionate or a glycinate. The complex is reminiscent of the carboxylate butyrylcholinesterase complexes observed in crystal structures of human butyrylcholinesterases from various sources, and demonstrates the remarkable ability of cholinesterases to stabilize covalent complexes with carboxylates. A very strong peak of density (10 σ) at covalent distance from the Cβ of the catalytic serine is present in the DmAChE/ZAI complex. This can be undoubtedly attributed to an iodine atom, suggesting an unanticipated iodo/hydroxyl exchange between Ser238 and the inhibitor, possibly driven by the intense X-ray irradiation. Finally, the binding of tacrine-derived inhibitors, such as ZA (1DX4) or the iodinated analog, ZAI (1QON) results in the appearance of an open channel that connects the base of the active-site gorge to the solvent. This channel, which arises due to the absence of the conserved tyrosine present in vertebrate cholinesterases, could be exploited to design inhibitors specific to insect cholinesterases. The present study demonstrates that updated processing of older diffraction images, and the re-refinement of older diffraction data, can produce valuable information that could not be detected in the original analysis, and strongly supports the preservation of the diffraction images in public data banks.
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(2020) Pacific Symposium on Biocomputing. 25, 2020, p. 149-158 Abstract
The concept that an unfolded protein could have biological function was proposed by Linus Pauling in 1940. The first experimental evidence for the existence of unfolded proteins, which are now often called intrinsically disordered proteins and regions (IDPs and IDRs), was published in the 1950s. From the 1950s to the 1990s, many additional articles describing IDPs or IDRs were published, and even several Nobel Prizes have been awarded for research on these proteins. The first computational biology/bioinformatics papers on these proteins were reported between 1998 and 2002, and these computational efforts rapidly increased shortly thereafter. In the late 1980s and mid-1990s, a few IDPs and IDRs involved with signaling and regulation were structurally characterized by nuclear magnet resonance (NMR), and these studies demonstrated a likely involvement of IDPs or IDRs in the associated biological regulation. Altogether, the computational biology/bioinformatics and NMR investigations were synergistic in stimulating the rapid increase in the research on IDPs and IDRs. Herein, we describe this history and recent developments in the IDP research arena. We also highlight a recent collection of papers on IDPs and IDRs that are driven by computational biology and bioinformatics efforts.
2019
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(2019) FEBS Journal. 286, 19, p. 3858-3873 Abstract
Interleukin 24 (IL-24) is a cytokine with the potential to be an effective treatment for autoimmune diseases and cancer. However, its instability and difficulties in its production have hampered detailed biological and biophysical studies. We approached the challenges of IL-24 production by using the PROSS algorithm to design more stable variants of IL-24. We used homology models built from the sequences and known structures of IL-20 and IL-19 and predicted and produced several extensively mutated IL-24 variants that were highly stable and produced in large yields; one of them was crystallized (IL-24B, PDB ID 6GG1; 3D Interactive at http://proteopedia.org/w/Journal: FEBS_Journal:1). The mutated variants, however, lost most of their binding capacity to the extracellular parts of cognate receptors. While the affinity to the receptor 2 (IL-20R2) was preserved, the variants lost affinity to IL-20R1 and IL-22R1 (shared receptors 1). Back engineering of the variants revealed that reintroduction of a single IL-24 wild-type residue (T198) to the patch interacting with receptors 1 restored 80% of the binding affinity and signaling capacity, accompanied by an acceptable drop in the protein stability by 9 degrees C. Multiple sequence alignment explains the stabilizing effect of the mutated residues in the IL-24 variants by their presence in the related and more stable cytokines IL-20 and IL-19. Our homology-based approach can enhance existing methods for protein engineering and represents a viable alternative to study and produce difficult proteins for which only in silico structural information is available, estimated as >40% of all important drug targets.
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(2019) F1000Research. 8, 1753. Abstract[All authors]
Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are now recognised as major determinants in cellular regulation. This white paper presents a roadmap for future e-infrastructure developments in the field of IDP research within the ELIXIR framework. The goal of these developments is to drive the creation of high-quality tools and resources to support the identification, analysis and functional characterisation of IDPs. The roadmap is the result of a workshop titled \u201cAn intrinsically disordered protein user community proposal for ELIXIR\u201d held at the University of Padua. The workshop, and further consultation with the members of the wider IDP community, identified the key priority areas for the roadmap including the development of standards for data annotation, storage and dissemination; integration of IDP data into the ELIXIR Core Data Resources; and the creation of benchmarking criteria for IDP-related software. Here, we discuss these areas of priority, how they can be implemented in cooperation with the ELIXIR platforms, and their connections to existing ELIXIR Communities and international consortia. The article provides a preliminary blueprint for an IDP Community in ELIXIR and is an appeal to identify and involve new stakeholders.
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(2019) Chemico-Biological Interactions. 310, 108715. Abstract
Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced breathing motions of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.
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(2019) Chemico-Biological Interactions. 309, 108699. Abstract
The crystal structures of truncated forms of cholinesterases provide good models for assessing the role of non-covalent interactions in dimer assembly in the absence of cross-linking disulfide bonds. These structures identify the four-helix bundle that serves as the interface for formation of acetylcholinesterase and butyrylcholinesterase dimers. Here we performed a theoretical comparison of the structural and energetic factors governing dimerization. This included identification of inter-subunit and infra-subunit hydrogen bonds and hydrophobic interactions, evaluation of solvent-accessible surfaces, and estimation of electrostatic contributions to dimerization. To reveal the contribution to dimerization of individual amino acids within the contact area, free energy perturbation alanine screening was performed. Markov state modelling shows that the loop between the alpha 13 and alpha 14 helices in BChE is unstable, and occupies 4 macro-states. The order of magnitude of mean first passage times between these macrostates is similar to 10(-8)s. Replica exchange molecular dynamics umbrella sampling calculations revealed that the free energy of human BChE dimerization is -15.5 kcal/mol, while that for human AChE is - 26.4 kcal/mol. Thus, the C-terminally truncated human butyrylcholinesterase dimer is substantially less stable than that of human acetylcholinesterase. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:1.
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(2019) European Journal of Medicinal Chemistry. 168, p. 58-77 Abstract[All authors]
Both cholinesterases (AChE and BChE) and kinases, such as GSK-3 alpha/beta, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3 alpha/beta (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5 nM for human acetylcholinesterase (hAChE) and 7 nM for GSK-3 alpha/beta. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp.
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(2019) Communications Chemistry. 2, 35. Abstract[All authors]
The development of polyphenols as drugs for Alzheimer's disease (AD) is thwarted by their meagre brain availability due to instability and poor druglikeness. Here we describe the successful development of stable, druglike polyphenolic analogues of the current AD drug rivastigmine, that have high apparent blood-brain barrier permeabilities and multifunctional properties for AD treatment. The compounds inhibit cholinesterases and amyloid beta (A beta) fibrillation, protect against A beta(42)-induced toxicity in vitro, and demonstrate efficacy in vivo in a transgenic Caenorhabditis elegans model expressing A beta(42), with potencies similar to rivastigmine and natural polyphenols. The results suggest that a tertiary amine substituent is amenable for developing water-soluble, membrane-permeable polyphenols, and its incorporation adjacent to a hydroxy group is favourable for intramolecular hydrogen bonding that facilitates membrane permeability. Carbamylation of one hydroxy group protects the polyphenols from degradation and mostly improves their membrane permeability. These design strategies may assist in the development of polyphenol-based drugs.
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(2019) European Journal of Medicinal Chemistry. 162, p. 234-248 Abstract[All authors]
Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously inhibit acetylcholinesterase and act as an agonist of the 5-HT4 receptor, which displays promising activities in vivo. Pharmacomodulation of donecopride allowed us to develop a novel series of indole derivatives possessing interesting in vitro activities toward AChE and the sigma(1) receptor. The crystal structures of complexes of the most promising compounds with Torpedo californica AChE were solved in order to further understand their mode of inhibition.
2018
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(2018) Proceedings Of The National Academy Of Sciences Of The United States Of America-Physical Sciences. 115, 52, p. 13270-13275 Abstract
The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryoelectron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 Å. Our structure reveals that the BChE tetramer is organized as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central lamellipodin-derived oligopeptide with a proline-rich attachment domain (PRAD) sequence that adopts a polyproline II helical conformation and runs antiparallel. The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases but may provide a promising template for designing other proteins with improved circulatory residence times.
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(2018) Journal of Medicinal Chemistry. 61, 17, p. 7630-7639 Abstract[All authors]
Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.
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(2018) Chemistry-A European Journal. 24, 38, p. 9675-9691 Abstract[All authors]
A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.
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(2018) FEBS Letters. 592, 12, p. 1909-1917 Abstract
iNEXT, 'Infrastructure for NMR, EM and X-rays for Translational research', is a EC-funded Horizon 2020 network of national infrastructures aiming to facilitate EC- supported, trans-national user access to several high-end structural biology infrastructures and to expand the use of structural biology technologies towards new research communities. iNEXT has developed different access routes offering cutting edge technologies and expertise, thus targeting the diverse needs of research groups that desire to perform X-ray crystallography, small-angle X-ray scattering, solution or solid-state NMR, single particle or cell tomography electron microscopy, biophysics, and imaging experiments. Specific Joint Research Activities aim at improving the user experience in ever advancing methods, and are focused on structure-based drug discovery, membrane protein characterization and structural cell biology initiatives.
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(2018) Journal of biomolecular structure & dynamics. 36, 9, p. 2331-2341 Abstract
Disordered protein chains and segments are fast becoming a major pathway for our understanding of biological function, especially in more evolved species. However, the standard definition of disordered residues: the inability to constrain them in X-ray derived structures, is not easily applied to NMR derived structures. We carry out a statistical comparison between proteins whose structure was resolved using NMR and using X-ray protocols. We start by establishing a connection between these two protocols for obtaining protein structure. We find a close statistical correspondence between NMR and X-ray structures if fluctuations inherent to the NMR protocol are taken into account. Intuitively this tends to lend support to the validity of both NMR and X-ray protocols in deriving biomolecular models that correspond to in vivo conditions. We then establish Lindemann-like parameters for NMR derived structures and examine what order/disorder cutoffs for these parameters are most consistent with X-ray data and how consistent are they. Finally, we find critical value of L = 4 for the best correspondence between X-ray and NMR derived order/disorder assignment, judged by maximizing the Matthews correlation, and a critical value L = 1.5 if a balance between false positive and false negative prediction is sought. We examine a few non-conforming cases, and examine the origin of the structure derived in X-ray. This study could help in assigning meaningful disorder from NMR experiments.
2017
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(2017) Molecules. 22, 8, 1324. Abstract
Functions of biomolecules, in particular enzymes, are usually modulated by structural fluctuations. This is especially the case in a gated diffusion-controlled reaction catalyzed by an enzyme such as acetylcholinesterase. The catalytic triad of acetylcholinesterase is located at the bottom of a long and narrow gorge, but it catalyzes the extremely rapid hydrolysis of the neurotransmitter, acetylcholine, with a reaction rate close to the diffusion-controlled limit. Computational modeling and simulation have produced considerable advances in exploring the dynamical and conformational properties of biomolecules, not only aiding in interpreting the experimental data, but also providing insights into the internal motions of the biomolecule at the atomic level. Given the remarkably high catalytic efficiency and the importance of acetylcholinesterase in drug development, great efforts have been made to understand the dynamics associated with its functions by use of various computational methods. Here, we present a comprehensive overview of recent computational studies on acetylcholinesterase, expanding our views of the enzyme from a microstate of a single structure to conformational ensembles, strengthening our understanding of the integration of structure, dynamics and function associated with the enzyme, and promoting the structure-based and/or mechanism-based design of new inhibitors for it.
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(2017) Journal of Neurochemistry. 142, p. 19-25 Abstract
This review focuses on several recent developments concerning structurefunction relationships in vertebrate acetylcholinesterase. These include studies on high-resolution structures of human acetylcholinesterase and its complexes; the first crystal structure of a snake venom acetylcholinesterase, in which open and closed states of the back door are visualized; a powerful algorithm for redesigning proteins for enhanced expression in prokaryotic systems, as applied to human acetylcholinesterase, which has hitherto been an intractable target; in situ implementation of click chemistry in crystalline acetylcholinesterase, which yields novel insights into the steric and dynamic changes involved in the reaction within the active-site gorge; and a study that demonstrates the effect of crystallization conditions on ligand alignment within a protein complex, in this case the methylene blueTorpedo californica acetylcholinesterase complex, which highlights the relevance of the precipitant employed to structure-based drug design. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. (Figure presented.).
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(2017) Protein Engineering, Design and Selection. 30, 4, p. 333-345 Abstract[All authors]
Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (k(cat)/K-M) of 5 x 10(6)M(-1) min(-1). However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of > 10(7) M-1 min(-1). Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a k(cat)/K-M >= 5 x 10(7)M(-1) min(-1) and Russian VX (RVX) with a k(cat)/K-M >= 10(7) M-1 min(-1). These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by > 5000-fold with VX and by > 17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at k(cat)/K-M > 5 x 10(7) M-1 min(-1)) and can thus serve as candidates for broadspectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.
2016
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(2016) Archives of Toxicology. 90, p. 2711-2724 Abstract[All authors]
The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (kcat/KM) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23s ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequenceactivity relationships along the pathway of PTEs laboratory evolution, we examined kcat/KM values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited kcat/KM values >107 M−1 min−1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequenceactivity relationships.
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(2016) Chemico-Biological Interactions. 259, p. 252-256 Abstract
Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme] pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme] pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s. c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in vivo was
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(2016) Toxicology Letters. 258, p. 198-206 Abstract
The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (similar to 2LD(50)). C23AL showed a C-max of 0.63 mu mol L (1) after i.o. and i.v. administration of 2 mg kg (1) providing a stable plasma profile up to 180 min experimental duration with 0.41 and 0.37 mu mol L (1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.
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(2016) Molecular Cell. 63, 2, p. 337-346 Abstract[All authors]
Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
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(2016) Protein Science. 25, 6, p. 1096-1114 Abstract
Structure-based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but
[All authors]
2015
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(2015) Journal of Molecular Biology. 427, 6 Part B, p. 1359-1374 Abstract
Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168-a key catalytic residue residing >15 angstrom from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.
2014
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(2014) Toxicology Letters. 231, 1, p. 45-54 Abstract[All authors]
The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(-) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (k(cat)/K-M = 5 x 10(6) M-1 min(-1)). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5 mg kg(-1) i.v.) 5 min after s.c. challenge with VX (similar to 2LD(50)) prevented systemic toxicity. A lower C23 dose (2 mg kg(-1)) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15 min post VX) with 5 mg kg(-1) C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(-)-VX enantiomer that correlates with the C23 blood levels and its k(cat)/K-M value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single postexposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication. (C) 2014 Elsevier Ireland Ltd. All rights
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(2014) Archives of Toxicology. 88, 6, p. 1257-1266 Abstract
The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (∼2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.
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(2014) Nucleic Acids Research. 42, D1, p. D326-D335 Abstract[All authors]
The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.
2013
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(2013) Israel Journal of Chemistry. 53, 3-4, p. 207-216 Abstract
Although Java does not run on some handheld devices, e.g., iPads and iPhones, JavaScript does. The development of JSmol, a JavaScript-only version of Jmol, is described, and its use in Proteopedia is demonstrated. A key aspect of JSmol is that it includes the full implementation of the entire set of Jmol functionalities, including file reading and writing, scripting, and rendering. The relative performances of Java-based Jmol and JavaScript-only JSmol are discussed. We can now confirm that the guiding principles of Java programming can be completely and relatively straightforwardly transformed directly into JavaScript, requiring no Java applet, and producing identical graphical results. JSmol is thus the first full-featured molecular viewer, and the first ever viewer for proteins, which can be utilized with an internet browser on handheld devices lacking Java. Since the MediaWiki features of Proteopedia have been modified to optionally use JSmol, the wealth of crowd-sourced content in Proteopedia is now directly available on such devices, without the need to download any additional applet.
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(2013) Chemico-Biological Interactions. 203, 1, p. 63-66 Abstract
The photosensitizer, methylene blue (MB), generates singlet oxygen ( 1O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ∼8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of 1O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.
[All authors] -
(2013) Journal of Molecular Biology. 425, 6, p. 1028-1038 Abstract
Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca2 +. We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-Å upward displacement towards the enzyme's surface of the catalytic Ca2 + in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca2 + mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions.
2012
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(2012) Macromolecular Crystallography. Carrondo M. A. & Spadon P.(eds.). p. 149-161 Abstract
Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other structures. Proteopedia ( http://www.proteopedia.org ) presents 3D biomolecule structures in a broadly accessible manner to a diverse scientific audience through easy-to-use molecular visualization tools integrated into a wiki environment that anyone with a user account can edit. We describe recent advances in the web resource in the areas of content and software. In terms of content, we describe a large growth in user-added content as well as improvements in automatically-generated content for all PDB entry pages in the resource. In terms of software, we describe new features ranging from the capability to create pages hidden from public view to the capability to export pages for offline viewing. New software features also include an improved file-handling system and availability of biological assemblies of protein structures alongside their asymmetric units.
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(2012) Protein Science. 21, 8, p. 1138-1152 Abstract
The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.
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(2012) Journal of Molecular Biology. 418, 3-4, p. 181-196 Abstract
The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.
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(2012) Chemistry and Biology. 19, 4, p. 456-466 Abstract
A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S P isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 10 7 M -1 min -1. Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) -4 in favor of the R P isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S P isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
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(2012) Biomolecular NMR Assignments. 6, 1, p. 15-18 Abstract
Neuroligins act as heterophilic adhesion molecules at neuronal synapses. Their cytoplasmic domains interact with synaptic scaffolding proteins, and have been shown to be intrinsically disordered. Here we report the backbone and side chain 1H, 13C and 15N resonance assignments for the cytoplasmic domain of human neuroligin 3.
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(2012) Acta Crystallographica Section F-Structural Biology And Crystallization Communications. 68, 3, p. 269-272 Abstract
The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 Å resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies.
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(2012) Nature Chemical Biology. 8, 3, p. 294-300 Abstract[All authors]
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (kcat/Km) of ∼104 M-1 s-1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the RP isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.
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(2012) Acta Crystallographica Section D: Biological Crystallography. 68, 1, p. 13-25 Abstract[All authors]
Β-Secretase (Β-site amyloid precursor protein-cleaving enzyme 1; BACE1) is a transmembrane aspartic protease that cleaves the Β-amyloid precursor protein en route to generation of the amyloid Β-peptide (AΒ) that is believed to be responsible for the Alzheimers disease amyloid cascade. It is thus a prime target for the development of inhibitors which may serve as drugs in the treatment and/or prevention of Alzheimers disease. In the following determination of the crystal structures of both apo and complexed BACE1, structural analysis of all crystal structures of BACE1 deposited in the PDB and molecular dynamics (MD) simulations of monomeric and dimeric BACE1 were used to study conformational changes in the active-site region of the enzyme. It was observed that a flap able to cover the active site is the most flexible region, adopting multiple conformational states in the various crystal structures. Both the presence or absence of an inhibitor within the active site and the crystal packing are shown to influence the flaps conformation. An open conformation of the flap is mostly observed in the apo structures, while direct hydrogen-bonding interaction between main-chain atoms of the flap and the inhibitor is a prerequisite for the flap to adopt a closed conformation in the crystal structures of complexes. Thus, a systematic study of the conformational flexibility of the enzyme may not only contribute to structure-based drug design of BACE1 inhibitors and of other targets with flexible conformations, but may also help to better understand the mechanistic events associated with the binding of substrates and inhibitors to the enzyme.
2011
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(2011) PROTEIN ENGINEERING DESIGN & SELECTION. 24, 10, p. 773-775 Abstract
Recently, two studies were published that examined the structure of the acid-β-glucosidase N370S mutant, the most common mutant that causes Gaucher disease. One study used the experimental tool of X-ray crystallography, and the other utilized molecular dynamics (MD). The two studies reinforced each other through the similarities in their findings, but each approach also added some unique information. Both studies report that the conformation of active site loop 3 changes, due to an altered hydrogen bonding network; however, the MD study produced additional data concerning the flexibility of loop 1 and the catalytic residues that are not observed in the other study.
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(2011) Toxicology Letters. 206, 1, p. 24-28 Abstract
An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its kcat/Km value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.
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(2011) Protein Science. 20, 8, p. 1285-1297 Abstract
Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.
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(2011) Journal of Structural Biology. 175, 2, p. 244-252 Abstract
Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other biomolecule structures. Created as a means for communicating biomolecule structures to a diverse scientific audience, Proteopedia (http://www.proteopedia.org) presents structural annotation in an intuitive, interactive format and allows members of the scientific community to easily contribute their own annotations. Here, we provide a status report on Proteopedia by describing advances in the web resource since its inception three and a half years ago, focusing on features of potential direct use to the scientific community. We discuss its progress as a collaborative 3D-encyclopedia of structures as well as its use as a complement to scientific publications and PowerPoint presentations. We also describe Proteopedia's use for 3D visualization in structure-related pedagogy.
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(2011) Journal of Structural Biology. 175, 2, p. 159-170 Abstract[All authors]
Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
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(2011) Protein Science. 20, 7, p. 1114-1118 Abstract
The transient opening of a backdoor in the active-site wall of acetylcholinesterase, one of nature's most rapid enzymes, has been suggested to contribute to the efficient traffic of substrates and products. A crystal structure of Torpedo californica acetylcholinesterase in complex with the peripheral-site inhibitor aflatoxin is now presented, in which a tyrosine at the bottom of the active-site gorge rotates to create a 3.4-angstrom wide exit channel. Molecular dynamics simulations show that the opening can be further enlarged by movement of Trp84. The crystallographic and molecular dynamics simulation data thus point to the interface between Tyr442 and Trp84 as the key element of a backdoor, whose opening permits rapid clearance of catalysis products from the active site. Furthermore, the crystal structure presented provides a novel template for rational design of inhibitors and reactivators, including anti-Alzheimer drugs and antidotes against organophosphate poisoning.
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(2011) Organic & Biomolecular Chemistry. 9, 11, p. 4160-4167 Abstract
Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of recombinant human acid β-glucosidase (β-glucocerebrosidase, GlcCerase) with amphiphilic bicyclic nojirimycin analogues of the sp2-iminosugar type. Attempts to co-crystallize GlcCerase with 5-N,6-O-[N-(n-octyl)iminomethylidene]nojirimycin (NOI-NJ) or with 5-N,6-S-[N-(n-octyl)iminomethylidene]-6-thionojirimycin (6S-NOI-NJ), two potent inhibitors of the enzyme with promising pharmacological chaperone activity for several Gaucher disease-associated mutations, were unsuccessful probably due to the formation of aggregates that increase the heterogeneity of the sample and affect nucleation and growth of crystals. Cyclomaltoheptaose (β-cyclodextrin, βCD) efficiently captures NOI-NJ and 6S-NOI-NJ in aqueous media to form inclusion complexes in which the lipophilic tail is accommodated in the hydrophobic cavity of the cyclooligosaccharide. The dissociation constant of the complex of the amphiphilic sp2-iminosugars with βCD is two orders of magnitude higher than that of the corresponding complex with GlcCerase, allowing the efficient transfer of the inhibitor from the βCD cavity to the GlcCerase active site. Enzyme-inhibitor complexes suitable for X-ray analysis were thus grown in the presence of βCD. In contrast to what was previously observed for the complex of GlcCerase with the more basic derivative, 6-amino-6-deoxy-5-N,6-N-[N-(n-octyl)iminomethylidene]nojirimycin (6N-NOI-NJ), the β-anomers of both NOI-NJ and 6S-NOI-NJ were seen in the active site, even though the α-anomer was exclusively detected both in aqueous solution and in the corresponding βCD:sp2-iminosugar complexes. Our results further suggest that cyclodextrin derivatives might serve as suitable delivery systems of amphiphilic glycosidase inhibitors in a biomedical context.
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(2011) Journal of Medicinal Chemistry. 54, 10, p. 3575-3580 Abstract
Selective estrogen receptor modulators, such as 17β-estradiol derivatives bound to metal complexes, have been synthesized as targeted probes for the diagnosis and treatment of breast cancer. Here, we report the detailed 3D structure of estrogen receptor α ligand-binding domain (ERα-LBD) bound with a novel estradiol-derived metal complex, estradiol-pyridine tetra acetate europium(III), at 2.6 Å resolution. This structure provides important information pertinent to the design of novel functional ERα targeted probes for clinical applications.
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(2011) Nature Chemical Biology. 7, 2, p. 120-125 Abstract[All authors]
Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >107 (M-1 min -1). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic Sp isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of Sp -cyclosarin by -105-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with kcat/KM ∼10 7 M-1 min-1. We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
2010
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(2010) Biophysical Journal. 99, 12, p. 4003-4011 Abstract
The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.
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(2010) Journal of Biological Chemistry. 285, 53, p. 42105-42114 Abstract
Gaucher disease is caused by the defective activity of the lysosomal hydrolase, glucosylceramidase. Although the x-ray structure of wild type glucosylceramidase has been resolved, little is known about the structural features of any of the >200 mutations. Various treatments for Gaucher disease are available, including enzyme replacement and chaperone therapies. The latter involves binding of competitive inhibitors at the active site to enable correct folding and transport of the mutant enzyme to the lysosome. We now use molecular dynamics, a set of structural analysis tools, and several statistical methods to determine the flexible behavior of the N370S Gaucher mutant at various pH values, with and without binding the chaperone, N-butyl- deoxynojirimycin. We focus on the effect of the chaperone on the whole protein, on the active site, and on three important structural loops, and we demonstrate how the chaperone modifies the behavior of N370S in such a way that it becomes more active at lysosomal pH. Our results suggest a mechanism whereby the binding of N-butyl-deoxynojirimycin helps target correctly folded glucosylceramidase to the lysosome, contributes to binding with saposin C, and explains the initiation of the substrate-enzyme complex. Such analysis provides a new framework for determination of the structure of other Gaucher disease mutants and suggests new approaches for rational drug design.
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(2010) Chemico-Biological Interactions. 187, 1-3, p. 10-22 Abstract
By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer's disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge.
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(2010) Chemico-Biological Interactions. 187, 1-3, p. 362-369 Abstract
Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic RP enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic SP enantiomer, and to determine accurately Km and kcat values for the individual isomers, two approaches were used to obtain the corresponding SP and RP isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the SP and RP isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The SP configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with SP/RP inhibition ratios of 10-100, whereas the RP isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the SP counterpart. Wt rePON1-induced RP/SP hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be 1000. The various SP enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents.
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(2010) Instrumental Analysis of Intrinsically Disordered Proteins. Longhi S. & N. Uversky V.(eds.). p. 695-704 Abstract
Since IDPs share physicochemical characteristics that differentiate them from globular proteins, the process of IDP purification can be highly efficient if one utilizes purification schemes that take advantage of these special characteristics. However, purification can be highly problematic when dealing with recombinant IDPs that are sensitive to the degradation machinery of the host cell in which they are being overexpressed. Herein, we survey some of the specialized procedures reported in the literature for purification of IDPs, elaborate on ways to stabilize IDPs in the course of purification, and focus on our experience in the purification of two highly protease-sensitive IDPs under denaturing conditions that inactivated the endogenous proteases of the host.
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(2010) Glycobiology. 20, 1, p. 24-32 Abstract
Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated T™ human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (Km and Vmax) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.
2009
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(2009) Journal of Medicinal Chemistry. 52, 23, p. 7593-7603 Abstract[All authors]
Organophosphate compounds (OP) are potent inhibitors of acetylcholinesterases (AChEs) and can cause lethal poisoning in humans. Inhibition of AChEs by the OP soman involves phosphonylation of the catalytic serine, and subsequent dealkylation produces a form known as the "aged" enzyme. The nonaged form can be reactivated to a certain extent by nucleophiles, such as pralidoxime (2-PAM), whereas aged forms of OP-inhibited AChEs are totally resistant to reactivation. Here, we solved the X-ray crystal structures of AChE from Torpedo californica (TcAChE) conjugated with soman before and after aging. The absolute configuration of the soman stereoisomer adduct in the nonaged conjugate is PSCR. A structural reorientation of the catalytic His440 side chain was observed during the aging process. Furthermore, the crystal structure of the ternary complex of the aged conjugate with 2-PAM revealed that the orientation of the oxime function does not permit nucleophilic attack on the phosphorus atom, thus providing a plausible explanation for its failure to reactivate the aged soman/AChE conjugate. Together, these three crystal structures provide an experimental basis for the design of new reactivators.
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(2009) PROTEIN ENGINEERING DESIGN & SELECTION. 22, 10, p. 641-648 Abstract
Predicting mutations that enhance protein-protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein-protein complexes.
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(2009) Biophysical Journal. 97, 8, p. 2316-2326 Abstract
Amalgam (Ama) is a secreted neuronal adhesion protein that contains three tandem immunoglobulin domains. It has both homophilic and heterophilic cell adhesion properties, and is required for axon guidance and fasciculation during early stages of Drosophila development. Here, we report its biophysical characterization and use small-angle x-ray scattering to determine its low-resolution structure in solution. The biophysical studies revealed that Ama forms dimers in solution, and that its secondary and tertiary structures are typical for the immunoglobulin superfamily. Ab initio and rigid-body modeling by small-angle x-ray scattering revealed a distinct V-shaped dimer in which the two monomer chains are aligned parallel to each other, with the dimerization interface being formed by domain 1. These data provide a structural basis for the dual adhesion characteristics of Ama. Thus, the dimeric structure explains its homophilic adhesion properties. Its V shape suggests a mechanism for its interaction with its receptor, the single-pass transmembrane adhesion protein neurotactin, in which each "arm" of Ama binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane.
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(2009) Biochemistry. 48, 28, p. 6644-6654 Abstract
Serum paraoxonases (PONs) are calcium-dependent lactonases with anti-atherogenic and detoxification functions. Here we describe the directed evolution and characterization of recombinant variants of serum paraoxonase PON3 that express in an active and soluble manner in Escherichia coli. These variants were obtained by combining family shuffling and phylogeny-based mutagenesis: the limited diversity of accessible, cloned PON3 genes was complemented by spiking the shuffling reaction with ancestor/consensus mutations, mutations to residues that comprise the consensus or appear in the predicted ancestors of the PON family. We screened the resulting libraries for PON3's lactonase activity while ensuring that the selected variants retained the substrate specificity of wild-type mammalian PON3s. The availability of highly stable, recombinant PON3 that is free of all other serum components enabled us to explore unknown biochemical features of PON3, including its binding to HDL particles, the effect of HDL on PON3's stability and enzymatic activity, and ex vivo tests of its anti-atherogenic properties. Overall, it appears that PON3 possesses properties very similar to those of PON1: the enzyme's lactonase activity is selectively stimulated by binding to apoAI-HDL, with a concomitant increase in its stability. PON3 also exhibits potentially antiatherogenic functions, although at levels lower than those of PON1.
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(2009) Journal of Medicinal Chemistry. 52, 8, p. 2543-2549 Abstract
A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and A beta peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 angstrom resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the "anionic" subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N(epsilon 2), which is hydrogen-bonded to Ser2000(gamma) in the native enzyme. The second nor-meptazinol binds at the peripheral "anionic" site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors.
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(2009) Protein Expression and Purification. 63, 2, p. 147-157 Abstract
Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH2-terminal His6Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high β-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with RH = 16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.
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(2009) Proteins-Structure Function And Bioinformatics. 77, SUPPL. 9, p. 50-65 Abstract
The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions. Analysis was based on the GDT-TS analysis, which has been used in previous CASP experiments, together with a newly developed method, the OK-Rank, as well as by visual inspection. There is no doubt that in recent years many obstacles have been removed on the long and elusive way to deciphering the protein-folding problem. Out of the 13 targets, six were predicted well by a number of groups. On the other hand, it must be stressed that for four targets, none of the models were judged to be satisfactory. Thus, for template free model prediction, as evaluated in this CASP, successes have been achieved for most targets; however, a great deal of research is still required, both in improving the existing methods and in development of new approaches.
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(2009) From Molecules to Medicines. Sussman J. L. & Spadon P.(eds.). p. 183-199 Abstract
Acetylcholinesterase and paraoxonase are important targets for treatment of degenerative diseases, Alzheimer's disease and atherosclerosis, respectively, both of which impose major burdens on the health care systems in Western society. Acetylcholinesterase is the target of lethal nerve agents, and paraoxonase is under consideration as a bioscavenger for their detoxification. Both are thus the subject of research and development in the context of nerve agent toxicology. The crystal structures of the two enzymes are described, and structure/function relationships are discussed in the context of drug development and of development of means of protection against chemical threats.
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(2009) From Molecules to Medicines. Sussman JL. & Spadon P.(eds.). p. 169-182 Abstract
To the crystallographer, solving a three-dimensional (3D) protein or molecular structure often times feels like the ultimate success, and surely it is. However, of utmost importance is the communication of the insights revealed by the 3D structure, especially those insights that relate structure to function. In order for these insights to reach their potential for guiding future research, they must reach biologists. The problem is that 3D structures are inherently complex and thus communicating insights about 3D structures to non-structural biologists can be difficult. To aid the structural biologist in this endeavor, we have created two useful tools. The first, eMovie, is a plugin for PyMOL that makes creating macromolecular animations much more simple. The second, Proteopedia, is a community-annotated 'wiki' web-resource that links descriptive text to 3D views of structures, resulting in intuitive communication of structural information.
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(2009) Proteins-Structure Function And Bioinformatics. 77, SUPPL. 9, p. 210-216 Abstract
The interest in intrinsically disordered proteins has greatly increased, as it has become clear that they are very widespread, especially in eukaryotic organisms. Functionally, they appear to play a significant role in the control of many cellular processes and signalling pathways and have been, also, associated with a number of diseases ranging from cancer to Alzheimer's. Thus, there is enormous interest in attempts to predict disordered regions in proteins solely from knowledge of their amino acid sequences. In this study, we assess the quality of predictions for 25 groups on predicting disordered regions in 122 target proteins. In addition, we suggest the need of a "knowledge- independent" measure that would enable one to normalize the results of the different CASP experiments and to determine whether the disorder prediction field had improved across the years.
2008
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(2008) Current Opinion in Structural Biology. 18, 6, p. 756-764 Abstract
The application of bioinformatics methodologies to proteins inherently lacking 3D structure has brought increased attention to these macromolecules. Here topics concerning these proteins are discussed, including their prediction from amino acid sequence, their enrichment in eukaryotes compared to prokaryotes, their more rapid evolution compared to structured proteins, their organization into specific groups, their structural preferences, their half-lives in cells, their contributions to signaling diversity (via high contents of multiple-partner binding sites, post-translational modifications, and alternative splicing), their distinct functional repertoire compared to that of structured proteins, and their involvement in diseases.
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(2008) Biological Chemistry. 389, 11, p. 1361-1369 Abstract
In mammalian cells, glucosylceramide (GlcCer), the simplest glycosphingolipid, is hydrolyzed by the lysosomal enzyme acid β-glucosidase (GlcCerase). In the human metabolic disorder Gaucher disease, GlcCerase activity is significantly decreased owing to one of approximately 200 mutations in the GlcCerase gene. The most common therapy for Gaucher disease is enzyme replacement therapy (ERT), in which patients are given intravenous injections of recombinant human GlcCerase; the Genzyme product Cerezyme® has been used clinically for more than 15 years and is administered to approximately 4000 patients worldwide. Here we review the crystal structure of Cerezyme® and other recombinant forms of GlcCerase, as well as of their complexes with covalent and non-covalent inhibitors. We also discuss the stability of Cerezyme®, which can be altered by modification of its N-glycan chains with possible implications for improved ERT in Gaucher disease.
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(2008) Biophysical Journal. 95, 5, p. 2500-2511 Abstract
The high aromatic content of the deep and narrow active-site gorge of acetylcholinesterase (AChE) is a remarkable feature of this enzyme. Here, we analyze conformational flexibility of the side chains of the 14 conserved aromatic residues in the active-site gorge of Torpedo californica AChE based on the 47 three-dimensional crystal structures available for the native enzyme, and for its complexes and conjugates, and on a 20-ns molecular dynamics (MD) trajectory of the native enzyme. The degree of flexibility of these 14 aromatic side chains is diverse. Although the side-chain conformations of F330 and W279 are both very flexible, the side-chain conformations of F120, W233, W432, Y70, Y121, F288, F290 and F331 appear to be fixed. Residues located on, or adjacent to, the Ω-loop (C67-C94), namely W84, Y130, Y442, and Y334, display different flexibilities in the MD simulations and in the crystal structures. An important outcome of our study is that the majority of the side-chain conformations observed in the 47 Torpedo californica AChE crystal structures are faithfully reproduced by the MD simulation on the native enzyme. Thus, the protein can assume these conformations even in the absence of the ligand that permitted their experimental detection. These observations are pertinent to structure-based drug design.
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(2008) Chemico-Biological Interactions. 175, 1-3, p. 3-10 Abstract
In accordance with its biological role, termination of neurotransmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase is one of nature's most efficient enzymes. Solution of its three-dimensional structure revealed that its active site is located at the bottom of a deep and narrow gorge. Such an architecture was unanticipated in view of its high turnover number. The present review examines how the highly specialized structure of acetylcholinesterase, with its sequestered active site, contributes to its catalytic efficacy, and discusses how the traffic of substrate and products to and from the active site is controlled.
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(2008) Proceedings of the National Academy of Sciences of the United States of America. 105, 33, p. 11742-11747 Abstract
Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the "off-switch" at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a "backdoor." The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
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(2008) GENOME BIOLOGY. 9, 8, R121. Abstract
Many scientists lack the background to fully utilize the wealth of solved three-dimensional biomacromolecule structures. Thus, a resource is needed to present structure/function information in a user-friendly manner to a broad scientific audience. Proteopedia http://www.proteopedia.org is an interactive, wiki web-resource whose pages have embedded three-dimensional structures surrounded by descriptive text containing hyperlinks that change the appearance (view, representations, colors, labels) of the adjacent three-dimensional structure to reflect the concept explained in the text.
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(2008) Biophysical Journal. 95, 4, p. 1928-1944 Abstract
Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In this study, we overexpress and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the Escherichia coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle x-ray scattering, circular dichroism, electron spin resonance, and nuclear magnetic resonance, we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.
[All authors] -
(2008) Journal of the American Chemical Society. 130, 25, p. 7856-7861 Abstract
Acetylcholinesterase plays a key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine with one of the highest known catalytic rate constants. Hydrolysis occurs in a narrow and deep gorge that contains two sites of ligand binding: A peripheral site, or P-site, near the gorge entrance that contributes to catalytic efficiency both by transiently trapping substrate molecules as they enter the gorge and by allosterically accelerating the transfer of the substrate acyl group to a serine hydroxyl in an acylation site or A-site at the base of the gorge. Thioflavin T is a useful reporter of ligand interactions with the A-site. It binds specifically to the P-site with fluorescence that is enhanced ∼1000-fold over that of unbound thioflavin T, and the enhanced fluorescence is quenched 1.5- to 4-fold when another ligand binds to the A-site in a ternary complex. To clarify the structural basis of this advantageous signal change, we here report the X-ray structure of the complex of thioflavin T with Torpedo californica acetylcholinesterase. The two aromatic rings in thioflavin T are coplanar and are packed snugly parallel to the aromatic side chains of Trp279, Tyr334, and Phe330. Overlays of this structure with the crystal structures of Torpedo californica acetylcholinesterase complexes with either edrophonium or m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone, two small aromatic ligands that bind specifically to the A-site, indicate that the phenyl side chain of Phe330 must rotate to sterically accommodate both thioflavin T and the A-site ligand in the ternary complex. This rotation may allow some relaxation of the strict coplanarity of the aromatic rings in the bound thioflavin T and result in partial quenching of its fluorescence.
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(2008) Proteins-Structure Function And Bioinformatics. 71, 2, p. 903-909 Abstract
Targeted turnover of proteins is a key element in the regulation of practically all basic cellular processes. The underlying physicochemical and/or sequential signals, however, are not fully understood. This issue is particularly pertinent in light of the recent recognition that intrinsically unstructured/disordered proteins, common in eukaryotic cells, are extremely susceptible to proteolytic degradation in vitro. The in vivo half-lives of proteins were determined recently in a high-throughput study encompassing the entire yeast proteome; here we examine whether these half-lives correlate with the presence of classical degradation motifs (PEST region, destruction-box, KEN-box, or the N-terminal residue) or with various physicochemical characteristics, such as the size of the protein, the degree of structural disorder, or the presence of low-complexity regions. Our principal finding is that, in general, the half-life of a protein does not depend on the presence of degradation signals within its sequence, even of ubiquitination sites, but correlates mainly with the length of its polypeptide chain and with various measures of structural disorder. Two distinct modes of involvement of disorder in degradation are proposed. Susceptibility to degradation of longer proteins, containing larger numbers of residues in conformational disorder, suggests an extensive function, whereby the effect of disorder can be ascribed to its mere physical presence. However, after normalization for protein length, the only signal that correlates with half-life is disorder, which indicates that it also acts in an intensive manner, that is, as a specific signal, perhaps in conjunction with the recognition of classical degradation motifs. The significance of correlation is rather low; thus protein degradation is not determined by a single characteristic, but is a multi-factorial process that shows large protein-to-protein variations. Protein disorder, nevertheless, plays a key signalling role in many cases.
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(2008) Protein Science. 17, 4, p. 601-605 Abstract
Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets. Published by Cold Spring Harbor Laboratory Press.
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(2008) Proteins-Structure Function And Bioinformatics. 70, 4, p. 1357-1366 Abstract
Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs.
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(2008) Nature Methods. 5, 2, p. 135-146 Abstract
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
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(2008) Methods in molecular biology (Clifton, N.J.). 426, p. 411-418 Abstract
Most high throughput structural proteomics centers use the sitting-drop method to obtain diffracting crystals for three-dimensional (3D) structure determination of biological macromolecules by x-ray crystallography. Although several robotic systems are available for dispensing the initial sitting-drop screening conditions, generally they are not used for optimization of crystallization conditions. This chapter describes a protocol for such automated systems, which permits easy construction of pH optimization grids using any desired fixed buffer set with varying ionic strengths directly dispensed into the crystallization plate.
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(2008) Journal of Applied Crystallography. 41, 5, p. 969-971 Abstract
Microbatch crystallization under oil is a powerful procedure for obtaining protein crystals. Using this method, aqueous protein solutions are dispensed under liquid oil, and water evaporates through the layer of oil, with a concomitant increase in the concentrations of both protein and precipitant until the nucleation point is reached. A technique is presented for regulating the rate of water evaporation, which permits fine tuning of the crystallization conditions as well as preventing complete desiccation of the drops in the microbatch crystallization trays.
2007
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(2007) Plant Biotechnology Journal. 5, 5, p. 579-590 Abstract[All authors]
Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme®) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme®. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme®. prGCD also displays a level of biological activity similar to that of Cerezyme® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.
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(2007) Journal of Biological Chemistry. 282, 39, p. 29052-29058 Abstract
Gaucher disease is caused by mutations in the gene encoding acid β-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca™, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2 Å resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars.
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(2007) Nucleic Acids Research. 35, SUPPL.2, p. W512-W521 Abstract
An Internet server at http://bip.weizmann.ac.il/dipol calculates the net charge, dipole moment and mean radius of any 3D protein structure or its constituent peptide chains, and displays the dipole vector superimposed on a ribbon backbone of the protein. The server can also display the angle between the dipole and a selected list of amino acid residues in the protein. When the net charges and dipole moments of ∼12 000 non-homologous PDB biological units (PISCES set), and their unique chains of length 50 residues or longer, were examined, the great majority of both charges and dipoles fell into a very narrow range of values, with long extended tails containing a few extreme outliers. In general, there is no obvious relation between a protein's charge or dipole moment and its structure or function, so that its electrostatic properties are highly specific to the particular protein, except that the majority of chains with very large positive charges or dipoles bind to ribosomes or interact with nucleic acids.
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(2007) Trends in Biochemical Sciences. 32, 5, p. 199-204 Abstract
The 3D structures of macromolecules are difficult to grasp and also to communicate. By their nature, movies or animations are particularly useful for highlighting key features by offering a 'guided tour' of structures and conformation changes. However, high-quality movies are rarely seen because they are currently difficult and time consuming to make. By adopting the traditional movie 'storyboard' concept, which gives guidance and direction to filming, eMovie makes the creation of lengthy molecular animations much easier. This tool is a plug-in for the open-source molecular graphics program PyMOL, and enables experts and novices alike to produce informative and high-quality molecular animations.
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(2007) Current Topics in Medicinal Chemistry. 7, 4, p. 375-387 Abstract
The naturally occurring alkaloid Huperzine A (HupA) is an acetylcholinesterase (AChE) inhibitor that has been used for centuries as a Chinese folk medicine in the context of its source plant Huperzia Serrata. The potency and relative safety of HupA rendered it a promising drug for the ameliorative treatment of Alzheimer's disease (AD) vis-à-vis the "cholinergic hypothesis" that attributes the cognitive decrements associated with AD to acetylcholine deficiency in the brain. However, recent evidence supports a neuroprotective role for HupA, suggesting that it could act as more than a mere palliative. Biochemical and crystallographic studies of AChE revealed two potential binding sites in the active-site gorge of AChE, one of which, the "peripheral anionic site" at the mouth of the gorge, was implicated in promoting aggregation of the beta amyloid (Aβ) peptide responsible for the neurodegenerative process in AD. This feature of AChE facilitated the development of dual-site binding HupA-based bivalent ligands, in hopes of concomitantly increasing AChE inhibition potency by utilizing the "chelate effect", and protecting neurons from Aβ toxicity. Crystal structures of AChE allowed detailed modeling and docking studies that were instrumental in enhancing the understanding of underlying principles of bivalent inhibitor-enzyme dynamics. This monograph reviews two categories of HupA-based bivalent ligands, in which HupA and HupA fragments serve as building blocks, with a focus on the recently solved crystallographic structures of Torpedo californica AChE in complex with such bifunctional agents. The advantages and drawbacks of such structured-based drug design, as well as species differences, are highlighted and discussed.
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(2007) Nature Structural & Molecular Biology. 14, 1, p. 3-4 Abstract
Over the next few years, structural proteomics will grapple with the problem of visualizing increasingly elaborate structures, from the atomic details of protein structures up to subcellular structures and the whole cell. A recent EU workshop addressed the question of what experimental and theoretical approaches, technologies and infrastructures this will demand.
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(2007) Acta Crystallographica Section D: Biological Crystallography. 63, 11, p. 1115-1128 Abstract[All authors]
Acetylcholinesterase plays a crucial role in nerve-impulse transmission at cholinergic synapses. The apparent paradox that it displays high turnover despite its active site being buried raises cogent questions as to how the traffic of substrates and products to and from the active site can occur so rapidly in such circumstances. Here, a kinetic crystallography strategy aimed at structurally addressing the issue of product traffic in acetylcholinesterase is presented, in which UV-laser-induced cleavage of a photolabile precursor of the enzymatic product analogue arsenocholine, 'caged' arsenocholine, is performed in a temperature-controlled X-ray crystallography regime. The 'caged' arsenocholine was shown to bind at both the active and peripheral sites of acetylcholinesterase. UV irradiation of a complex with acetylcholinesterase during a brief temperature excursion from 100 K to room temperature is most likely to have resulted in a decrease in occupancy by the caged compound. Microspectrophotometric experiments showed that the caged compound had indeed been photocleaved. It is proposed that a fraction of the arsenocholine molecules released within the crystal had been expelled from both the active and the peripheral sites. Partial q-weighted difference refinement revealed a relative movement of the two domains in acetylcholinesterase after photolysis and the room-temperature excursion, resulting in an increase in the active-site gorge volume of 30% and 35% in monomers A and B of the asymmetric unit, respectively. Moreover, an alternative route to the active-site gorge of the enzyme appeared to open. This structural characterization of acetylcholinesterase 'at work' is consistent with the idea that choline exits from the enzyme after catalysis either via the gorge or via an alternative 'backdoor' trajectory.
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Examination of the tRNA adaptation index as a predictor of protein expression levels(2007) Systems Biology And Regulatory Genomics. 4023, p. 107-118 Abstract
Phenotypic differences between closely-related species may arise from differential expression regimes, rather than different gene complements. Knowledge of cellular protein levels across a species sample would thus be useful for the inference of the genes underlying such phenotypic differences. dos Reis et al [1] recently proposed the tRNA Adaptation Index to score the optimality of a coding sequence with respect to a species' cellular tRNA pools. As a preliminary step towards a multi-species analysis that would utilize this index, we examine in this paper its performance in predicting protein expression levels in the yeast S. cerevisiae and find that it likely predicts maximal potential levels of proteins. We also show that tAI profiles of genes across species carry functional information regarding the interactions between proteins.
2006
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(2006) Acta Crystallographica Section D-Biological Crystallography. 62, p. 1458-1465 Abstract
Gaucher disease is caused by mutations in the gene encoding acid-beta-glucosidase. A recombinant form of this enzyme, Cerezyme((R)), is used to treat Gaucher disease patients by 'enzyme- replacement therapy'. Crystals of Cerezyme((R)) after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 angstrom resolution [Dvir et al. (2003), EMBO Rep. 4, 704 - 709]. The crystal structure of unmodified Cerezyme1 is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme((R)) is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-beta-glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes.
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(2006) Acta Crystallographica Section D: Biological Crystallography. 62, 10, p. 1260-1266 Abstract
Not all proteins form well defined three-dimensional structures in their native states. Some amino-acid sequences appear to strongly favour the disordered state, whereas some can apparently transition between disordered and ordered states under the influence of changes in the biological environment, thereby playing an important role in processes such as signalling. Although important biologically, for the structural biologist disordered regions of proteins can be disastrous even preventing successful structure determination. The accurate prediction of disorder is therefore important, not least for directing the design of expression constructs so as to maximize the chances of successful structure determination. Such design criteria have become integral to the construct-design strategies of laboratories within the Structural Proteomics In Europe (SPINE) consortium. This paper assesses the current state of the art in disorder prediction in terms of prediction reliability and considers how best to use these methods to guide construct design. Finally, it presents a brief discussion as to how methods of prediction might be improved in the future.
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(2006) Acta Crystallographica Section D: Biological Crystallography. 62, 10, p. 1208-1217 Abstract
The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ∼170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.
[All authors] -
(2006) Acta Crystallographica Section D: Biological Crystallography. 62, 10, p. 1232-1242 Abstract
Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.
[All authors] -
(2006) ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY. 62, 10, p. 1184-1195 Abstract
SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.
[All authors] -
(2006) Acta Crystallographica Section D: Biological Crystallography. 62, 10, p. 1137-1149 Abstract
This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future.
[All authors] -
(2006) Acta Crystallographica Section D: Biological Crystallography. 62, 10, p. 1114-1124 Abstract
The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
[All authors] -
(2006) Journal of Medicinal Chemistry. 49, 18, p. 5491-5500 Abstract[All authors]
The X-ray crystal structures were solved for complexes with Torpedo californica acetylcholinesterase of two bivalent tacrine derivative compounds in which the two tacrine rings were separated by 5- and 7-carbon spacers. The derivative with the 7-carbon spacer spans the length of the active-site gorge, making sandwich interactions with aromatic residues both in the catalytic anionic site (Trp84 and Phe330) at the bottom of the gorge and at the peripheral anionic site near its mouth (Tyr70 and Trp279). The derivative with the 5-carbon spacer interacts in a similar manner at the bottom of the gorge, but the shorter tether precludes a sandwich interaction at the peripheral anionic site. Although the upper tacrine group does interact with Trp279, it displaces the phenyl residue of Phe331, thus causing a major rearrangement in the Trp279-Ser291 loop. The ability of this inhibitor to induce large-scale structural changes in the active-site gorge of acetylcholinesterase has significant implications for structure-based drug design because such conformational changes in the target enzyme are difficult to predict and to model.
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(2006) Gaucher Disease. Futerman A. H. & Zimran A.(eds.). p. 85-96 Abstract
Acid β -glucosidase (EC 3.2.1.45, D-glucosyl- N -acylsphingosine glucohydrolase, glucocerebrosidase, GlcCerase), the enzyme defective in Gaucher disease, is a peripheral lysosomal membrane protein that hydrolyzes the β -glucosyl linkage of glucosylceramide (GlcCer). GlcCerase requires the coordinate action of saposin C and negatively charged lipids for maximal activity. 1,2 Enzyme replacement therapy (ERT) with Cerezyme ®, a recombinant human GlcCerase, 3 is the main treatment for Type 1 Gaucher disease. Although attempts at structural prediction had been made earlier, 4,5 the lack of an experimental 3-D structure of GlcCerase hampered attempts to establish its catalytic mechanism and to analyze the structural relationships between the mutations, levels of residual enzyme activity, and disease severity. The recent determination in our laboratories of the x-ray structures of GlcCerase at 2.0 Å resolution 6 and, subsequently, of a conjugate with an irreversible inhibitor, 7 conduritol B epoxide (CBE), has paved the way for detailed structural analysis of GlcCerase. In this chapter we will review the two structures, and discuss the insights that they provide that may help in designing second-generation GlcCerases for ERT.
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(2006) EMBO Journal. 25, 12, p. 2746-2756 Abstract
Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.
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(2006) Structure. 14, 4, p. 629-630 Abstract
In this issue of Structure, Nanao and Ravelli (2006) describe the use of UV-induced radiation damage (UV-RIP) to solve the phase problem for proteins, employing single-wavelength X-ray radiation, without the need for derivatization. This should also permit data collection for many proteins on home sources, without travel to a synchrotron.
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(2006) Lecture Notes in Control and Information Sciences. p. 863-868 Abstract
Prophages are integrated viral genomes in bacteria. Prophages are distinct from other genomic segments encoding virulence factors that have been acquired by horizontal gene transfer events. A database for prophages (http://bicmku.in:8082/prophagedb http://ispc.weizmann.ac.il/prophagedb) has been constructed with data available from literature reports. To date other than bacteriophage corner stone genes based iterative searches, no other exhaustive approach unique for identifying prophage elements is available. Here we report detection of prophages based on proteomic signature comparison using a prophage proteome as reference set. This method was tested with using the database and then extended over newly sequenced bacterial genomes with no reported prophages. The approach of using similarity of proteins over a given region helped identify twenty putative prophage regions in nine different bacterial genomes.
2005
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(2005) Journal Of Physical Chemistry B. 109, 49, p. 23730-23738 Abstract
The unbinding process of E2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) leaving from the long active site gorge of Torpedo califomica acetylcholinesterase (TcAChE) was studied by using steered molecular dynamics (SMD) simulations on a nanosecond scale with different velocities, and unbinding force profiles were obtained. Different from the unbinding of other AChE inhibitors, such as Huperzine A that undergoes the greatest barrier located at the bottleneck of the gorge, the major resistance preventing E2020 from leaving the gorge is from the peripheral anionic site where E2020 interacts intensively with several aromatic residues (e.g., Tyr70, Tyr121, and Trp279) through its benzene ring and forms a strong direct hydrogen bond and a water bridge with Ser286 via its O24. These interactions cause the largest rupture force, ~550 pN. It was found that the rotatable bonds of the piperidine ring to the benzene ring and dimethoxyindanone facilitate E2020 to pass the bottleneck through continuous conformation change by rotating those bonds to avoid serious conflict with Tyr121 and Phe330. The aromatic residues lining the gorge wall are the major components contributing to hydrophobic interactions between E2020 and TcAChE. Remarkably, these aromatic residues, acting in three groups as "sender" and "receiver", compose a "conveyer belt" for E2020 entering and leaving the TcAChE gorge.
[All authors] -
(2005) Chemico-Biological Interactions. 157-158, p. 247-252 Abstract[All authors]
CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer prodrug that has been approved for the treatment of colon cancer. It is a member of the camptothecin class of drugs and activation to the active metabolite SN-38, is mediated by carboxylesterases (CE). SN-38 is a potent topoisomerase I poison and is highly effective at killing human tumor cells, with IC50 values in the low nM range. However, upon high dose administration of CPT-11 to cancer patients, a cholinergic syndrome is observed, that can be rapidly ameliorated by atropine. This suggests a direct interaction of the drug or its metabolites with acetylcholinesterase (AChE). Kinetic studies indicated that CPT-11 was primarily responsible for AChE inhibition with the 4-piperidinopiperidine moiety, the major determinant in the loss of enzyme activity. Structural analogs of 4-piperidinopiperidine however, did not inhibit AChE, including a benzyl piperazine derivate of CPT-11. These results suggest that novel anticancer drugs could be synthesized that do not inhibit AChE, or alternatively, that novel AChE inhibitors could be designed based around the camptothecin scaffold.
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(2005) Chemico-Biological Interactions. 157-158, p. 153-157 Abstract
The anticancer prodrug CPT-11 is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. The 2.6 Å resolution crystal structure of its complex with Torpedo californica acetylcholinesterase (TcAChE) demonstrates that CPT-11 binds to TcAChE and spans its gorge similarly to the Alzheimer drug, Aricept. The crystal structure clearly reveals the interactions, which contribute to the inhibitory action of CPT-11. Modeling of the complexes of CPT-11 with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug, shows how binding to either of the two enzymes yields a productive substrate-enzyme complex.
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(2005) Acta Crystallographica Section D: Biological Crystallography. 61, 10, p. 1426-1431 Abstract
A crystallization screening process is presented that was developed for a small academic laboratory. Its underlying concept is to combine sparse-matrix screening with systematic screening in a minimum number of crystallization conditions. The sparse-matrix screen is the cherry-picked combination of conditions from the Joint Center for Structural Genomics (JCSG) extended using conditions from other screens. Its aim is to maximize the coverage of crystallization parameter space with no redundancy. The systematic screen, a pH-, anion- and cation-testing (PACT) screen, aims to decouple the components of each condition and to provide information about the protein, even in the absence of crystals, rather than cover a wide crystallization space. This screening strategy is combined with nanolitre-volume dispensing hardware and a small but practical experiment-tracking system. The screens have been tested both at the NKI and in other laboratories and it is concluded that they provide a useful minimal screening strategy.
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(2005) Acta Crystallographica Section D: Biological Crystallography. 61, 10, p. 1364-1372 Abstract
The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.
[All authors] -
(2005) Bioinformatics. 21, 16, p. 3435-3438 Abstract
Summary: An easy-to-use, versatile and freely available graphic web server, FoldIndex
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(2005) Journal of the American Chemical Society. 127, 31, p. 11029-11036 Abstract
Recently, alkylene-linked heterodimers of tacrine (1) and 5-amino-5,6,7,8-tetrahydroquinolinone (2, hupyridone) were shown to exhibit higher acetylcholinesterase (AChE) inhibition than either monomeric 1 or 2. Such inhibitors are potential drug candidates for ameliorating the cognitive decrements in early Alzheimer patients. In an attempt to understand the inhibition mechanism of one such dimer, (RS)-(±)-N-9-(1,2,3,4- tetrahydroacridinyl)-N-5-[5,6,7,8-tetrahydro-2(1H) -quinolinonyl]-1,10-diaminodecane[(RS)-(±)-3] bisoxalate, the racemate was soaked in trigonal Torpedo californica AChE (TcAChE) crystals, and the X-ray structure of the resulting complex was solved to 2.30 A ̊ resolution. Its structure revealed the 1 unit bound to the "anionic" subsite of the active site, near the bottom of the active-site gorge, as seen for the 1/TcAChE complex. Interestingly, only the (R)-enantiomer of the 2 unit was seen in the peripheral "anionic" site (PAS) at the top of the gorge, and was hydrogen-bonded to the side chains of residues belonging to an adjacent, symmetry-related AChE molecule covering the gorge entrance. When the same racemate was soaked in orthorhombic crystals of TcAChE, in which the entrance to the gorge is more exposed, the crystal structure of the corresponding complex revealed no substantial enantiomeric selectivity. This observation suggests that the apparent enantiomeric selectivity of trigonal crystals of TcAChE for (R)-3 is mainly due to crystal packing, resulting in preferential binding of one enantiomeric inhibitor both to its "host" enzyme and to its neighbor in the asymmetric unit, rather than to steric constraints imposed by the geometry of the active-site gorge.
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(2005) Acta Crystallographica Section D: Biological Crystallography. 61, 6, p. 671-678 Abstract
Structural genomics aims at the establishment of a universal protein-fold dictionary through systematic structure determination either by NMR or X-ray crystallography. In order to catch up with the explosive amount of protein sequence data, the structural biology laboratories are spurred to increase the speed of the structure-determination process. To achieve this goal, high-throughput robotic approaches are increasingly used in all the steps leading from cloning to data collection and even structure interpretation is becoming more and more automatic. The progress made in these areas has begun to have a significant impact on the more 'classical' structural biology laboratories, dramatically increasing the number of individual experiments. This automation creates the need for efficient data management. Here, a new piece of software, HalX, designed as an 'electronic lab book' that aims at (i) storage and (ii) easy access and use of all experimental data is presented. This should lead to much improved management and tracking of structural genomics experimental data.
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(2005) Journal of Biological Chemistry. 280, 25, p. 23815-23819 Abstract
Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-β-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 Å resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep. 4, 704-709) and have now solved the structure of GlcCerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu 340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.
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(2005) Molecular Pharmacology. 67, 6, p. 1874-1881 Abstract
The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-] carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.
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(2005) Current Opinion in Pharmacology. 5, 3 SPEC. ISS., p. 293-302 Abstract
The synaptic enzyme acetylcholinesterase (AChE) terminates transmission at cholinergic synapses by rapidly hydrolysing acetylcholine. It is anchored within the synaptic cleft by a highly specialized anchoring device in which catalytic subunit tetramers assemble around a polyproline II helix. AChE is the target of nerve agents, insecticides and therapeutic drugs, in particular the first generation of anti-Alzheimer drugs. Both target-guided synthesis and structure-based drug design have been used effectively to obtain potent anticholinesterase agents. In addition, AChE is believed to play 'non-classical' roles in addition to its 'classical' role in terminating synaptic transmission (e.g. as an adhesion protein). It also accelerates assembly of Aβ into amyloid fibrils. Both of these actions involve the so-called 'peripheral' anionic site at the entrance to the active-site gorge. Novel anticholinesterases are targeted against this site, rather than against the active site at the bottom of the gorge.
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(2005) Proceedings of the National Academy of Sciences of the United States of America. 102, 21, p. 7493-7498 Abstract
Protein molecular adaptation to drastically shifting salinities was studied in dCA II, an α-type carbonic anhydrase (EC 4.2.1.1) from the exceptionally salt-tolerant unicellular green alga Dunaliella salina. The salt-inducible, extracellular dCA II is highly salt-tolerant and thus differs from its mesophilic homologs. The crystal structure of dCA II, determined at 1.86-Å resolution, is globally similar to other α-type carbonic anhydrases except for two extended α-helices and an added Na-binding loop. Its unusual electrostatic properties include a uniformly negative surface electrostatic potential of lower magnitude than that observed in the highly acidic halophilic proteins and an exceptionally low positive potential at a site adjoining the catalytic Zn2+ compared with mesophilic homologs. The halotolerant dCA II also differs from typical halophilic proteins in retaining conformational stability and solubility in low to high salt concentrations. The crucial role of electrostatic features in dCA II halotolerance is strongly supported by the ability to predict the unanticipated halotolerance of the murine CA XIV isozyme, which was confirmed biochemically. A proposal for the functional significance of the halotolerance of CA XIV in the kidney is presented.
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(2005) PROTEIN ENGINEERING DESIGN & SELECTION. 18, 2, p. 103-110 Abstract
Intrinsically unstructured proteins (IUPs) or IUP-like regions often play key roles in controlling processes ranging from transcription to the cell cycle. In silico such proteins can be identified by their sequence properties; they have low hydrophobicity and high net charge. In this study, we applied the FoldIndex (http://bioportal.weizmann.ac.il/fldbin/findex) program to analyze human G protein-coupled receptors and compared them with membrane proteins of known structure and with IUPs. We show that human G protein-coupled receptor (GPCR) extramembranous domains include long (>50 residues) disordered segments, unlike membrane proteins of known structure. The predicted disorder occurred primarily in the N-terminal, C-terminal and third intracellular domain regions: 55, 69 and 56% of the human GPCRs were disordered in these regions, respectively. This increased flexibility may therefore be critical for GPCR function. Surprisingly, however, the kinds of residues used in GPCR unstructured regions were different than in hitherto-identified IUPs. The GPCR third intracellular loop domains contain very high percentages of Arg, Lys and His residues, especially Arg, but the percentage of Glu, Asp and Pro is no higher than in folded proteins. We propose that this has structural and functional consequences.
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(2005) Adaptation To Life At High Salt Concentrations In Archaea, Bacteria, And Eukarya. Dordrecht, Netherlands: . Vol. 9. p. 503-515 (trueCellular Origin, Life in Extreme Habitats and Astrobiology). Abstract
Keywords: HALOPHILIC MALATE-DEHYDROGENASE; HALOFERAX-VOLCANII; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; STABILITY; ARCHAEON; ADAPTATION; BACTERIUM; OVEREXPRESSION; PURIFICATION
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(2005) Current Alzheimer Research. 2, 2, p. 207-218 Abstract
Acetylcholinesterase (AChE) plays a crucial physiological role in termination of impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine. In addition, it was implicated in amyloid plaque formation, a hallmark of Alzheimer's disease (AD), and most of the drugs used in AD treatment are AChE inhibitors. Thus ACHE is an obvious candidate gene for pharmacogenetic study of AD treatment. However, AChE is a highly conserved molecule, and only a few naturally occurring genetic polymorphisms have been reported in the human gene. The goals of this study were to make a systematic effort to identify natural single nucleotide polymorphisms (SNPs) in the human ACHE gene, and to reveal their population specific architecture. To this end, the genomic coding sequences for AChE of 96 unrelated control individuals from three distinct ethnic groups, African Americans, Ashkenazi Jews and Israeli Arabs, were analyzed. Thirteen ACHE SNPs were identified, ten of which are newly described, and five of which should produce amino-acid substitutions (Arg34Gln, Gly57Arg, Glu344Gly, His353Asn and Pro592Arg). Population frequencies of 11 of the 13 SNPs were established in four different populations, African Americans, Ashkenazi Jews, Sephardic Jews and Israeli Arabs; 17 haplotypes and 5 ethno-specific alleles were identified, and a cladogram of ACHE haplotypes was constructed. Among the SNPs resulting in an amino-acid substitution, three are within the mature protein, mapping on its external surface; they are thus unlikely to affect its catalytic properties, yet could have antigenic consequences or affect putative protein-protein interactions. Furthermore, the newly identified SNPs open the door to a study of the possible association of AChE with deleterious phenotypes - such as adverse drug responses to AChE inhibitors employed in treatment of AD patients and hypersensitivity to pesticides.
2004
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(2004) Journal of the American Chemical Society. 126, 47, p. 15405-15411 Abstract
Bifunctional derivatives of the alkaloid galanthamine, designed to interact with both the active site of the enzyme acetylcholinesterase (AChE) and its peripheral cation binding site, have been assayed with Torpedo californica AChE (TcAChE), and the three-dimensional structures of their complexes with the enzyme have been solved by X-ray crystallography. Differences were noted between the IC50 values obtained for TcAChE and those for Electrophorus etectricus AChE. These differences are ascribed to sequence differences in one or two residues lining the active-site gorge of the enzyme. The binding of one of the inhibitors disrupts the native conformation of one wall of the gorge, formed by the loop Trp279-Phe290. It is proposed that flexibility of this loop may permit the binding of inhibitors such as galanthamine, which are too bulky to penetrate the narrow neck of the gorge formed by Tyr121 and Phe330 as seen in the crystal structure.
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(2004) EMBO Journal. 23, 22, p. 4394-4405 Abstract
Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an ∼4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChET tetramer.
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(2004) Journal of Physical Chemistry A. 108, 43, p. 9400-9405 Abstract
The π-cation-π interaction between a cation or a cationic group and several aromatic residues, although rather prevalent in biological systems, has not been studied theoretically. The ab initio MP2 calculations were carried out on the systems composed of TMA with two aromatic rings, viz. benzene, pyrrole, or indole, to explore how a cation or a cationic group interacts simultaneously with two aromatic residues in proteins or nucleic acids. The calculated results on π-TMA-π complexes revealed additivities of both the geometries and the binding energies relative to cation-π complexes. The preferred structure of such a complex can be constructed by superimposing the corresponding TMA-π complexes via the cation. The binding energies of the π-TMA-π sandwiches are the sums of the two corresponding TMA-π systems. The contribution of electron correlation to the overall binding energy is estimated to be at least 50%, with dispersion serving as the main component of the electron correlation interaction. Similar to geometrical and energetic additivity, the additivities in BSSE and ΔZPE were also found. Therefore, our finding provides a convenient and effective way to construct π-TMA-π sandwiches and to estimate their binding energies. Morokuma decomposition analysis on the binding energy indicated that the electrostatic, charge transfer, and polarity interactions drive the binding of TMA with aromatics, whereas the exchange repulsion and high order coupling always obstruct the TMA approaching aromatics. Charge-transfer happens to some extent during the complexation of TMA with aromatics, and the transferred NPA atomic charges and charge-transfer energies are almost same in different complexes of TMA-π or π-TMA-π. The interaction between the 2 aromatics in the sandwich π-TMA-π complexes is negligible because of their long interaction distances. All this information should be helpful in studying such interactions in biological systems.
[All authors] -
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(2004) Nature Structural & Molecular Biology. 11, 5, p. 412-419 Abstract[All authors]
Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 Å. PON1 is a six-bladed β-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.
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(2004) Trends in Pharmacological Sciences. 25, 3, p. 147-151 Abstract
Gaucher disease, an inherited metabolic disorder caused by mutations in the gene encoding acid-β-glucosidase (GlcCerase), is a multi-system disease whose manifestations include anemia, thrombocytopenia, hepatosplenomegaly, bone pathology and, in some cases, neurological signs. Enzyme replacement therapy (ERT) using recombinant GlcCerase (Cerezyme®) alleviates many disease symptoms and is used by ~3000 patients worldwide, and substrate-reduction therapy (SRT) using the glycolipid synthesis inhibitor N-butyldeoxynojirimycin [NB-DNJ (Zavesca®)] has been approved recently for patients for whom ERT is unsuitable. It is our opinion that a multiplicity of treatment strategies is required for the management of Gaucher disease. In this article, we discuss the pros and cons of currently available treatments, and suggest complementary therapies arising from the determination of the X-ray structure of Cerezyme® and from delineation of secondary biochemical pathways affected in Gaucher disease.
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(2004) PROTEIN ENGINEERING DESIGN & SELECTION. 17, 2, p. 191-200 Abstract
Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea. An unusual, internally duplicated, 60 kDa α-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities. It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability. Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO2 hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration. Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl. Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I. Hence dCA I is a uniquely salt-tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions. Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.
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(2004) Human Mutation. 24, 5, p. 408-416 Abstract
Acetylcholinesterase (AChE) plays a crucial physiological role in termination of impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine. It is a highly conserved molecule, and only a few naturally occurring genetic polymorphisms have been reported in the human gene. The goal of the present study was to make a systematic effort to identify natural single nucleotide polymorphisms (SNPs) in the human ACHE gene. To this end, the genomic coding sequences for acetylcholinesterase of 96 unrelated control individuals from three distinct ethnic groups were analyzed. A total of 13 ACHE SNPs were identified, 10 of which are newly described, and five that should produce amino acid substitutions [c.101G>A (p.Arg34Gln), c.169G>A (p.Gly57Arg), c.1031A>G (p.Glu344Gly), c.1057C>A (p.His353Asn), and c.1775C>G (p.Pro592Arg)]. Population frequencies of 11 of the 13 SNPs were established in four different populations: African Americans, Ashkenazi Jews, Sephardic Jews, and Israeli Arabs; 15 haplotypes and five ethnospecific alleles were identified. The low number of SNPs identified until now in the ACHE gene is ascribed to technical hurdles arising from the high GC content and the presence of numerous repeat sequences, and does not reflect its intrinsic heterozygosity. Among the SNPs resulting in an amino acid substitution, three are within the mature protein, mapping on its external surface: they are thus unlikely to affect its catalytic properties, yet could have antigenic consequences or affect putative protein-protein interactions. Furthermore, the newly identified SNPs open the door to a study of the possible association of AChE with deleterious phenotypes - such as adverse drug responses to AChE inhibitors employed in treatment of Alzheimer patients and hypersensitivity to pesticides.
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(2004) Proteins-Structure Function And Genetics. 54, 1, p. 20-40 Abstract
Availability of complete genome sequences allows in-depth comparison of single-residue and oligopeptide compositions of the corresponding proteomes. We have used principal component analysis (PCA) to study the landscape of compositional motifs across more than 70 genera from all three superkingdoms. Unexpectedly, the first two principal components clearly differentiate archaea, eubacteria, and eukaryota from each other. In particular, we contrast compositional patterns typical of the three superkingdoms and characterize differences between species and phyla, as well as among patterns shared by all compositional proteomic signatures. These species-specific patterns may even extend to subsets of the entire proteome, such as proteins pertaining to individual yeast chromosomes. We identify factors that affect compositional signatures, such as living habitat, and detect strong eukaryotic preference for homopeptides and palindromic tripeptides. We further detect oligopeptides that are either universally over- or underabundant across the whole proteomic landscape, as well as oligopeptides whose over- or underabundance is phylum- or species-specific. Finally, we report that species composition signatures preserve evolutionary memory, providing a new method to compare phylogenetic relationships among species that avoids problems of sequence alignment and ortholog detection.
2003
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(2003) Proteins-Structure Function And Genetics. 53, 3, p. 758-767 Abstract
Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-1H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).
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(2003) Journal of the American Chemical Society. 125, 37, p. 11340-11349 Abstract
The entering and leaving processes of Huperzine A (HupA) binding with the long active-site gorge of Torpedo californica acetylcholinesterase (TcAChE) have been investigated by using steered molecular dynamics simulations. The analysis of the force required along the pathway shows that it is easier for HupA to bind to the active site of AChE than to disassociate from it, which for the first time interprets at the atomic level the previous experimental result that unbinding process of HupA is much slower than its binding process to AChE. The direct hydrogen bonds, water bridges, and hydrophobic interactions were analyzed during two steered molecular dynamics (SMD) simulations. Break of the direct hydrogen bond needs a great pulling force. The steric hindrance of bottleneck might be the most important factor to produce the maximal rupture force for HupA to leave the binding site but it has a little effect on the binding process of HupA with AChE. Residue Asp72 forms a lot of water bridges with HupA leaving and entering the AChE binding gorge, acting as a clamp to take out HupA from or put HupA into the active site. The flip of the peptide bond between Gly117 and Gly118 has been detected during both the conventional MD and SMD simulations. The simulation results indicate that this flip phenomenon could be an intrinsic property of AChE and the Gly117-Gly118 peptide bond in both HupA bound and unbound AChE structures tends to adopt the native enzyme structure. At last, in a vacuum the rupture force is increased up to 1500 pN while in water solution the greatest rupture force is about 800 pN, which means water molecules in the binding gorge act as lubricant to facilitate HupA entering or leaving the binding gorge.
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(2003) EMBO Reports. 4, 7, p. 704-709 Abstract
Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ∼200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non-neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α 8 TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.
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(2003) Acta Crystallographica Section D: Structural Biology. 59, 6, p. 1084-1086 Abstract
An extracellular α-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations. To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method. The crystals belonged to space group P21, with unit-cell parameters a = 47.0, b = 119.9, c= 58.5 Å, β = 94.2°. Data from a single crystal were collected to 2.4 Å resolution under cryogenic conditions (120 K) using an R-AXIS IV++ detector mounted on a Rigaku RU-H3R rotating-anode generator. The asymmetric unit contains two molecules of the protein, which corresponds to VM = 2.65 Å3 Da-1 and a solvent content of 52.7%.
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(2003) Protein Expression and Purification. 28, 1, p. 151-157 Abstract
A 60-kDa, salt-inducible, internally duplicated α-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.
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(2003) Biopolymers. 68, 3, p. 395-406 Abstract
In order to visualize and appreciate conformational changes between homologous three-dimensional (3D) protein structures or protein/inhibitor complexes, we have developed a user-friendly morphing procedure. It enabled us to detect coordinated conformational changes not easily discernible by analytic methods or by comparison of static images. This procedure was applied to comparison of native Torpedo californica acetylcholinesterase and of complexes with reversible inhibitors and conjugates with covalent inhibitors. It was likewise shown to be valuable for the visualization of conformational differences between acetylcholinesterases from different species. The procedure involves generation, in Cartesian space, of 25 interpolated intermediate structures between the initial and final 3D structures, which then serve as the individual frames in a QuickTime movie.
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(2003) Journal of Molecular Neuroscience. 20, 3, p. 369-383 Abstract
The structure of Torpedo californica acetylcholinesterase is examined in complex with several inhibitors that are either in use or under development for treating Alzheimer's disease. The noncovalent inhibitors vary greatly in their structures and bind to different sites of the enzyme, offering many different starting points for future drug design.
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(2003) Myasthenia Gravis And Related Disorders: Biomchemical Basis For Disease Of The Neuromuscular Junction. 998, p. 93-100 Abstract
Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) α-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by α-bungarotoxin (α-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and α-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to α-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of α-BTX to the AChR with an IC50 of 2 nM. The solution and crystal structures of complexes of α-BTX with HAP were solved, demonstrating that the HAP fits snugly to α-BTX and adopts a β-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that α-BTX wraps around the receptor binding-site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand-binding site. Our proposed model explains the strong antagonistic activity of α-BTX and accommodates much of the biochemical data on the mode of interaction of α-BTX with the AChR.
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(2003) Journal of the American Chemical Society. 125, 2, p. 363-373 Abstract
Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 Å resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE ∼6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds ∼3- and ∼2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.
2002
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(2002) Biophysical Chemistry. 100, 1-3, p. 293-305 Abstract
α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC50 of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.
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(2002) Journal of Synchrotron Radiation. 9, 6, p. 342-346 Abstract
Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by ∼0.7 Å upon X-ray irradiation as seen in a series of nine data sets collected on a single crystal. Simulation of the same bond suggests elongation by a similar value if a disulfide-radical anion is formed by trapping an electron. The absorption spectrum of a crystal irradiated under similar conditions shows a peak at ∼400 nm, which in aqueous solution has been attributed to disulfide radicals. The results suggest that the formation of disulfide radicals in protein crystals owing to X-ray irradiation can be observed experimentally, both by structural means and by absorption spectroscopy.
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(2002) Acta Crystallographica Section D: Biological Crystallography. 58, 10 I, p. 1765-1771 Abstract
The X-ray crystal structure of Torpedo californica acetylcholinesterase (TcAChE) complexed with BW284C51 {CO[-CH2CH2-pC6H4-N(CH 3)2 (CH2-CH=CH2)]2} is described and compared with the complexes of two other active-site gorge-spanning inhibitors, decamethonium and E2020. The inhibitor was soaked into TcAChE crystals in the trigonal space group P3121, yielding a complex which diffracted to 2.85 Å resolution. The structure was refined to an R factor of 19.0% and an Rfree of 23.4%; the final model contains the protein, inhibitor, 132 water molecules and three carbohydrate moieties. BW284C51 binds similarly to decamethonium and E2020, with its two phenyl and quaternary amino end-groups complexed to Trp84 in the catalytic site and to Trp279 in the peripheral binding site, and its central carbonyl group hydrogen bonded very weakly to Tyr121. Possible reasons for decamethonium's weaker binding are considered. The relative strength of binding of bisquaternary inhibitors to acetylcholinesterase and the effect of several mutations of the enzyme are discussed in the context of the respective X-ray structures of their complexes with the enzyme.
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(2002) Biochemistry. 41, 35, p. 10810-10818 Abstract
Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 μM, respectively, compared to 0.18 μM for (-)-huperzine A. The x-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 Å resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through π-π stacking and through van der Waals or C-H···π interactions with Trp84 and Phe330. Since their α-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H···π interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.
[All authors] -
(2002) Biochemistry. 41, 9, p. 2970-2981 Abstract
Huprine X is a novel acetylcholinesterase (ACHE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 Å resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.
[All authors] -
(2002) Biochemistry. 41, 11, p. 3555-3564 Abstract
Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (ki = 2.0 M-1 min-1), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is > 1600-fold higher (ki = 3300 M-1 min-1). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (ki = 9 × 104 and 5 × 105 M-1 min-1, respectively). Spontaneous reactivation of all four conjugates is very slow, with
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(2002) Journal of Molecular Biology. 320, 4, p. 721-725 Abstract
The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 Å resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH2 groups of the inhibitor, which are engaged in C-H···π interactions that structurally mimic the cation-π interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H···O and C-H···π types.
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(2002) Journal of Physical Chemistry A. 106, 1, p. 157-164 Abstract
The theoretical investigation of tetramethylammonium (TMA)-imidazole and TMA-furan complexes has been performed to justify the preferred structure of the cation-aromatic complexes predicated by the analysis of molecular electrostatic potential (MEP) maps of isolated aromatic systems. Such maps have been shown to be helpful in predicting the cation binding sites in cation-aromatic complexes. Our computational results show that a large part of the binding energies in the systems studied are contributed by the classical cation-π interaction. However, the optimized structure obtained in the MP2 method might be different from that obtained by the DFT method due to the influence of dispersion forces. Dispersion forces have been found to be important in the systems studied, increasing the binding energies by ∼7% and 20% for the TMA-imidazole and TMA-furan systems, respectively.
[All authors]
2001
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(2001) Chemical Physics Letters. 349, 1-2, p. 113-122 Abstract
Calculations on alkaline earth metal ion-benzene complexes were performed using the density-functional theory (DFT) B3LYP and ab initio MP2 methods. They showed that the interaction is very strong, of the order of magnitude of a normal chemical bond. Electrostatic interaction is not the dominant component, and both charge transfer and induction make a significant contribution. Analysis of molecular orbital interactions indicated that binding of the alkaline earth metal ions to benzene may be attributed to s-π and p-π interactions, which are significantly stronger than those between alkali cations and benzene.
[All authors] -
(2001) Neuron. 32, 2, p. 265-275 Abstract[All authors]
We have determined the crystal structure at 1.8 Å resolution of a complex of α-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the α subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a β hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.
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(2001) Journal of Medicinal Chemistry. 44, 20, p. 3203-3215 Abstract
Chimeras of tacrine and m-(N,N,N-Trimethylammonio)trifluoroacetophenone (1) were designed as novel, reversible inhibitors of acetylcholinesterase. On the basis of the X-ray structure of the apoenzyme, a molecular modeling study determined the favored attachment positions on the 4-aminoquinoline ring (position 3 and the 4-amino nitrogen) and the favored lengths of a polymethylene link between the two moieties (respectively 5-6 and 4-5 sp3 atoms). Seven compounds matching these criteria were synthesized, and their inhibitory potencies were determined to be in the low nanomolar range. Activity data for close analogues lacking some of the postulated key features showed that our predictions were correct. In addition, a subsequent crystal structure of acetylcholinesterase complexed with the most active compound 27 was in good agreement with our model. The design strategy is therefore validated and can now be developed further.
[All authors] -
(2001) Journal of Physical Chemistry A. 105, 22, p. 5431-5437 Abstract
A detailed theoretical investigation of the tetramethylammonium(TMA)-benzene and TMA-pyrrole complexes has been performed to obtain the interaction properties of TMA with aromatics. Diffuse functions have been found to be important in the computational studies of these noncovalent complexes. Adding diffuse functions to the basis set decreases the binding energy by about 10% for the TMA-aromatic systems. Dispersion interactions in the TMA-aromatic systems are very important. They enhance the binding interactions between the TMA and the aromatic ring systems by about 0.5 kcal·mol-1 per interacting atomic pair, which is in agreement with the estimates of Rappé and Bernstein.1 Also, for the TMA-pyrrole complex, the presence of the dispersion interaction leads to a dramatic change in the optimized structure. Because B3LYP cannot handle properly the dispersion in the calculation, use of the Møller-Plesset second-order perturbation or other sophisticated methods should be considered in computational studies of cation-π interactions in systems containing nonsymmetric dispersion interacted atomic pairs. The orbital interaction is unimportant in the TMA-aromatic interaction according to the detailed analysis of the molecular orbitals. The TMA-aromatic interactions basically come from the typical cation-π interaction and the dispersion interaction. Because the electron density in the Π56 aromatic system of pyrrole is larger than that in the Π66 system of benzene, the π electron cloud on pyrrole is more easily polarized under the influence of cations, which may lead to a relatively stronger cation-π interaction in the TMA-pyrrole complex than in the TMA-benzene complex.
[All authors] -
(2001) Journal of Physical Chemistry A. 105, 8, p. 1326-1333 Abstract
To consider whether existing molecular force fields can adequately reproduce cation-π interactions without adding special interaction terms, theoretical calculations with geometry optimization were performed on three configurations of tetramethylammonium (TMA) interacting via one, two, or three W-methyl groups with a benzene ring, by use of density-functional theory (DFT) methods B3LYP/6-31G* and B3LYP/6-311G**, ab initio method MP2/6-31G*, and molecular mechanic methods EFF, Tinker's Amber and MM3. Only the first configuration was found to be stable from the DFT and MP2 results, and its geometry was found to be highly flexible. ESP CHELPG charges estimated from the DFT and MP2 calculations were used to modify the atomic charges of the force fields employed in the molecular mechanics calculations to improve agreement with the BSSE-corrected binding energies deduced from the DFT and MP2 results. After this modification, the molecular mechanics results were found to be in good agreement with those obtained by DFT and MP2, without a requirement to add any additional terms to the force fields. This was confirmed by comparing the energy profiles of the complex as benzene was moved away from TMA in 0.2 Å intervals. Hence it is possible to use existing force fields to represent cation-π interactions by a simple adjustment of certain partial atomic charge parameters. In this context, we discuss the high flexibility of the cation-π interactions in the framework of molecular recognition in biological systems.
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(2001) Acta Crystallographica Section D: Biological Crystallography. 57, 4, p. 566-573 Abstract
Solvent behaviour in flash-cooled protein crystals was assessed by monitoring the expansion of unit-cell parameters as a function of temperature. Solvent in large channels undergoes a glass transition and crystallizes at 155 K.
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(2001) Protein Science. 10, 10, p. 1953-1961 Abstract
The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired - at least locally - sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.
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(2001) Acta Crystallographica Section D: Biological Crystallography. 57, 9, p. 1348-1350 Abstract
Histochemical methods are employed to detect and localize a wide range of enzymes. Even though protein crystallographers do not commonly use this technique, the extensively used colorimetric reaction of Karnovsky was successfully adapted for easy and quick identification of acetylcholinesterase crystals. The method relies on the reduction of ferricyanide to ferrocyanide by thiocholine, released from acetylthiocholine by enzymatic hydrolysis, followed by formation of a cupric ferrocyanide precipitate, and allows rapid differentiation between salt and enzyme crystals and between native and inhibited crystals of the enzyme.
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The Protein Data Bank at Brookhaven(2001) International Tables for Crystallography. Vol. F, p. 649-656 [All authors]
2000
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(2000) Journal of Physical Chemistry A. 104, 42, p. 9573-9580 Abstract
DFT/B3LYP calculations were carried out on complexes formed by NH4+ with aromatics, viz. benzene, phenol, pyrrole, imidazole, pyridine, indole, furane, and thiophene, to characterize the forces involved in such interactions and to gain further insight into the nature and diversity of cation-aromatic interactions. Such calculations may provide valuable information for understanding molecular recognition in biological systems and for force-field development. B3LYP/6-31G** optimization on 35 initial structures resulted in 11 different finally optimized geometries, which could be divided into three types: NH4+-π complexes, protonated heterocyclic-NH3 hydrogen bond complexes, and heterocyclic-NH4+ hydrogen bond complexes. For NH4+-π complexes, NH4+ always tilts toward the carbon-carbon bond rather than toward the heteroatom or the carbon-heteroatom bond. The calculated CHelpG charges suggest that the charge distribution of a free heterocyclic may be used to predict the geometry of its complex. Charge population and electrostatic interaction estimations show that the NH4+-π interaction has the largest nonelectrostatic interaction fraction (approximately 47%) of the total binding energy, while the NH4+-aromatic hydrogen bond interaction has the largest electrostatic fraction (approximately 90%). A good correlation between binding energy and electrostatic interaction in the NH4+-π complexes is found, which shows that nonelectrostatic interaction is important for cation-π binding. The results calculated with basis sets from 6-31G to 6-311++G(2df, 2dp) show that ΔEcorr and ΔHcorr do not require a basis-set superposition error (BSSE) correction, in view of experimental error, if a larger basis set is used in the calculation. The calculated ΔHcorr values for the NH4+-C6H6 complex with different basis sets suggest that the experimental ΔH may be overestimated.
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(2000) Journal of Molecular Biology. 296, 2, p. 713-735 Abstract
Buried water molecules and the water molecules in the active-site gorge are analyzed for five crystal structures of acetylcholinesterase from Torpedo californica in the resolution range 2.2-2.5 Å (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identified, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the five structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central β-sheet structure. The active-site gorge of the native enzyme is filled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in the native enzyme. Possible roles of the buried water molecules are discussed, including their possible action as a lubricant to allow large-amplitude fluctuations of the loop structures forming the gorge wall. Such fluctuations are required to facilitate traffic of substrate, products and water molecules to and from the active-site. Because of their poor coordination, the gorge water molecules can be considered as 'activated' as compared to bulk water. This should allow their easy displacement by incoming substrate. The relatively loose packing of the gorge water molecules leaves numerous small voids, and more efficient space-filling by substrates and inhibitors may be a major driving force of ligand binding. (C) 2000 Academic Press.
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(2000) Drug Development Research. 50, 3-4, p. 573-583 Abstract
The structure of Torpedo californica acetylcholinesterase (AChE) was examined in complex with several inhibitors which are either in use or under development for treatment of Alzheimer's disease. The inhibitors vary greatly in their structures, offering differing starting points for future drug design. The structure of T. californica AChE is also compared to the recently solved structure of Drosophila melanogaster AChE, the first invertebrate enzyme to have its structure determined. The structure of D. melanogaster AChE complexed with a potential insecticide is described. (C) 2000 Wiley-Liss, Inc.
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(2000) Acta Crystallographica Section D: Biological Crystallography. 56, 11, p. 1385-1394 Abstract[All authors]
Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 Å resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 Å resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.
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Inactivation studies of acetylcholinesterase with phenylmethylsulfonyl fluoride(2000) Molecular Pharmacology. 57, 6, p. 1243-1248 Abstract
Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsufonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Tropedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290l, and the double mutant T. californica F288L/F290V (all mutants given T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290l had increased rate constants (~14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290l mouse mutant AChEs, representing an increase of ~4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.
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(2000) Acta Crystallographica Section D: Biological Crystallography. 56, 7, p. 828-841 Abstract
This paper describes the design and full implementation of a new concept in data deposition and validation: AutoDep (copyright Brookhaven Science Associates LLC). AutoDep changes the traditional procedure for data acceptance and validation of the primary databases into an interactive depositor-driven operation which almost eliminates the delay between the acceptance of the data and its public release. The system takes full advantage of the knowledge and expertise of the experimenters, rather than relying on the database curators for the complete and accurate description of the structural experiment and its results. AutoDep, developed by the Protein Data Bank at Brookhaven National Laboratory (BNL) as a flexible and portable system, has already been adopted by other primary databases and implemented on different platforms/operating systems. AutoDep was introduced at BNL in 1996 [see Manning (1996), Protein Data Bank Quart. Newslett. 77, 2 (ftp://ftp.rcsb.org/pub/pdb/doc/newsletters/bnl/newsletter96jul/newslttr.txt) ; Manning (1996), Protein Data Bank Quart. Newslett. 78, 2 (ftp://ftp.rcsb.org/pub/pdb/doc/newsletters/bnl/newsletter96oct/newslttr.txt) ].
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(2000) Proceedings of the National Academy of Sciences of the United States of America. 97, 2, p. 623-628 Abstract[All authors]
Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative 'weak links' in a given biological macromolecule, which may be of structural and functional significance.
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(2000) Protein Science. 9, 6, p. 1063-1072 Abstract
We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9- acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine- all-three at 2.7 Å resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 Å from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica ACHE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is ~50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.
1999
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(1999) FEBS Letters. 463, 3, p. 321-326 Abstract
(-)-Galanthamine (GAL), an alkaloid from the flower, the common snowdrop (Galanthus nivalis), shows anticholinesterase activity. This property has made GAL the target of research as to its effectiveness in the treatment of Alzheimer's disease. We have solved the X-ray crystal structure of GAL bound in the active site of Torpedo californica acetylcholinesterase (TcAChE) to 2.3 Å resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline-binding site (Trp-84) and the acyl-binding pocket (Phe-288, Phe-290). The tertiary amine group of GAL does not interact closely with Trp-84; rather, the double bond of its cyclohexene ring stacks against the indole ring. The tertiary amine appears to make a non-conventional hydrogen bond, via its N-methyl group, to Asp-72, near the top of the gorge. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu-199. The relatively tight binding of GAL to TcAChE appears to arise from a number of moderate to weak interactions with the protein, coupled to a low entropy cost for binding due to the rigid nature of the inhibitor.
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(1999) Biophysical Journal. 77, 5, p. 2430-2450 Abstract
We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced ~10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.
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Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase(1999) Molecular Pharmacology. 55, 6, p. 982-992 Abstract
Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis Showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.
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(1999) Biochemistry. 38, 22, p. 7032-7039 Abstract[All authors]
Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (ACHE) and then may undergo an internal dealkylation reaction (called 'aging') to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (satin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 Å resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.
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(1999) Chemico-Biological Interactions. 119-120, p. 43-52 Abstract
Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements. Copyright (C) 1999 Elsevier Science Ireland Ltd.
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(1999) Structure. 7, 3, p. 297-307 Abstract
Background: Several cholinesterase inhibitors are either being utilized for symptomatic treatment of Alzheimer's disease or are in advanced clinical trials. E2020, marketed as Aricept®, is a member of a large family of N- benzylpiperidine-based acetylcholinesterase (ACHE) inhibitors developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies, prior to elucidation of the three-dimensional structure of Torpedo californica AChE (TcAChE). E2020 significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for ACHE, binding to both electric eel and mouse AChE in the nanomolar range. Results: Our experimental structure of the E2020-TcAChE complex pinpoints specific interactions responsible for the high affinity and selectivity demonstrated previously. It shows that E2020 has a unique orientation along the active-site gorge, extending from the anionic subsite of the active site, at the bottom, to the peripheral anionic site, at the top, via aromatic stacking interactions with conserved aromatic amino acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole', but only indirectly via solvent molecules. Conclusions: Our study shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.
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(1999) Genetica. 106, 1-2, p. 149-158 Abstract
The protein data bank (PDB), at Brookhaven National Laboratory, is a database containing information on experimentally determined three-dimensional structures of proteins, nucleic acids, and other biological macromolecules, with approximately 9000 entries. The PDB has a 27-year history of service to a global community of researchers, educators, and students in a wide variety of scientific disciplines. Data are easily submitted via PDB's WWW-based tool AutoDep, in either PDB or mmCIF format, and are most conveniently examined via PDB's WWW-based tool 3DB Browser. Collaborative centers have been, and continue to be, established worldwide to assist in data deposition, archiving, and distribution.
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1998
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(1998) Acta Crystallographica Section D: Biological Crystallography. 54, 6 I, p. 1078-1084 Abstract
The Protein Data Bank (PDB) at Brookhaven National Laboratory, is a database containing experimentally determined three-dimensional structures of proteins, nucleic acids and other biological macromolecules, with approximately 8000 entries. Data are easily submitted via PDB's WWW-based tool AutoDep, in either mmCIF or PDB format, and are most conveniently examined via PDB's WWW-based tool 3DB Browser.
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(1998) Protein Engineering. 11, 6, p. 415-420 Abstract
The concept of an electrostatic motif on the surface of biological macromolecules as a definite topographical pattern of electrostatic potentials in three-dimensional space, provides a powerful tool for identification of functionally important regions on the surface of structurally related macromolecules. Using this approach, we identify a functional region common to cholinesterases (ChEs) and to a set of neural cell-adhesion proteins that have been suggested to be structurally related to cholinesterases due to their high sequence similarity, but lacking the key catalytically active serine. Quantitative analysis of the electrostatic surface potential in the area surrounding the entrance to the active site of acetylcholinesterase, and in the analogous zone for the ChE-like domain of the adhesion proteins reveals very good correlation. These findings, examined in the context of previous evidence involving this same region in a possible cell-recognition function for ChEs, leads us to define a class of adhesion proteins which we have named 'electrotactins'.
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Re: letter to the editor regarding deposition of 3D structural studies of biological macromolecules.(1998) Biochemical and Biophysical Research Communications. 245, 3, p. 946 Abstract
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Immediate release of crystallographic data: A proposal [3](1998) Science. 279, 5349, p. 306-307 Abstract
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Three-dimensional structure of a complex of E2020 with acetylcholinesterase from Torpedo californica(1998) Journal Of Physiology-Paris. 92, 3-4, p. 191-194 Abstract
The 3D structure of a complex of the anti-Alzheimer drug, E2020, also known as Aricept®, with Torpedo californica acetylcholinesterase is reported. The X-ray structure, at 2.5 Å resolution, shows that the elongated E2020 molecule spans the entire length of the active-site gorge of the enzyme. It thus interacts with both the 'anionic' subsite, at the bottom of the gorge, and with the peripheral anionic site, near its entrance, via aromatic stacking interactions with conserved aromatic residues. It does not interact directly with either the catalytic triad or with the 'oxyanion hole'. Although E2020 is a chiral molecule, and both the S and R enantiomers have similar affinity for the enzyme, only the R enantiomer is bound within the active-site gorge when the racemate is soaked into the crystal. The selectivity of E2020 for acetylcholinesterase, relative to butyrylcholinesterase, can be ascribed primarily to its interactions with Trp279 and Phe330, which are absent in the latter.
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3D structure at 2.7A resolution of native and E202Q mutant human acetylcholinesterase complexed with fasciculin-II(1998) Structure And Function Of Cholinesterases And Related Proteins. p. 323-326 Abstract[All authors]
Keywords: PHOSPHYLATED HUMAN ACETYLCHOLINESTERASE; RECOMBINANT HUMAN ACETYLCHOLINESTERASE; ALZHEIMERS-DISEASE; CRYSTAL-STRUCTURE; ACTIVE-CENTER; INHIBITION; REFINEMENT; EXPRESSION; SECRETION; RESIDUES
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(1998) Acta Crystallographica Section D: Biological Crystallography. 54, 6 PART II, p. 1359-1366 Abstract
Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from monochromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding.
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Crystallographic studies on complexes of acetylcholinesterase with the natural cholinesterase inhibitors fasciculin and huperzine A(1998) Progress In Alzheimer'S And Parkinson'S Diseases. 49, p. 523-530 Abstract
Keywords: Behavioral Sciences; Clinical Neurology; Psychiatry
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Quaternary structure of tetrameric acetylcholinesterase(1998) Structure And Function Of Cholinesterases And Related Proteins. p. 351-+ Abstract
Keywords: Biochemistry & Molecular Biology
1997
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(1997) JOURNAL OF MOLECULAR GRAPHICS & MODELLING. 15, 5, p. 318-327 Abstract
The electrostatic potentials for the three-dimensional structures of cholinesterases from various species were calculated, using the Delphi algorithm, on the basis of the Poisson-Boltzmann equation. We used structures for Torpedo californica and mouse acetylcholinesterase, and built homology models of the human, Bungarus fasciatus, and Drosophila melanogaster acetylcholinsterases and human butyrylcholinesterase. All these structures reveal a negative external surface potential, in the area around the entrance to the active-site gorge, that becomes more negative as the rim of the gorge is approached. Moreover, in all cases, the potential becomes increasingly more negative along the central axis running down the gorge, and is largest at the base of the gorge, near the active site. Ten key acidic residues conserved in the sequence alignments of AChE from various species, both in the surface area near the entrance of the active-site gorge and at its base, appear to be primarily responsible for these potentials. The potentials are highly correlated among the structures examined, down to sequence identities as low as 35%. This indicates that they are a conserved property of the cholinesterase family, could serve to attract the positively charged substrate into and down the gorge to the active site, and may play other roles important for cholinesterase function.
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(1997) Macromolecular Crystallography Part B. Vol. 277. p. 556-571 Abstract
This chapter discusses the Protein Data Bank (PDB) archives of three-dimensional (3D) macromolecular structures. Several pieces of information related to an entry are archived by the PDB. In addition to the coordinate entry file, the PDB stores files related to the experiment such as structure factors, nuclear Overhaüser effect (NOE) restraints, and lists of chemical shifts. Also archived are auxiliary files used in structure analysis and refinement such as X-PLOR parameter and topology files. Currently, the archives are managed as a set of individual files, and each entry may have several associated files. The PDB is in the process of building a relational database, 3Dbase that will replace the current data management and access system. A description of 3DBase, including an outline of the way users can access its contents is provided in the chapter. Coordinate entries in the PDB are stored in separate files, each of which reports the results of an experiment or analysis that elucidates the structure of proteins, nucleic acids, polysaccharides, and other biological macromolecules. Although most of the data are generated from single crystal X-ray diffraction studies, a growing number of PDB entries are from nuclear magnetic resonance (NMR) studies.
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(1997) Nature Structural Biology. 4, 1, p. 57-63 Abstract
(-)-Huperzine A (HupA) is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. Its action has been attributed to its ability to strongly inhibit acetylcholinesterase (ACHE). The crystal structure of the complex of AChE with optically pure HupA at 2.5 Å, resolution shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. This structure is compared to the native structure of AChE devoid of any inhibitor as determined to the same resolution. An analysis of the affinities of structural analogues of HupA, correlated with their interactions with the protein, shows the importance of individual hydrophobic interactions between HupA and aromatic residues in the active-site gorge of AChE.
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How three-fingered snake toxins recognise their targets - Acetylcholinesterase-fasciculin complex, a case study(1997) Theoretical And Computational Methods In Genome Research. p. 303-315 Abstract
Three-fingered toxins from snake venoms constitute a family of 6-8kDa proteins, which can be divided into a number of groups with widely varying targets. The most studied of these are the a-neurotoxins which have long been used for the purification and characterisation of nicotinic acetylcholine receptors. Another group are the fasciculins, which specifically inhibit another essential synaptic component, the enzyme acetylcholinesterase. The structure of acetylcholinesterase is described, with emphasis on its unique features of relevance to its interaction with fasciculin. Furthermore, all three-fingered toxins whose structures have been determined are compared at both the sequence and structural level. With the additional use of a multiple sequence alignment of all known three-fingered toxins, residues of fasciculins which were expected to be important in the interaction with acetylcholinesterase were identified. The recently determined three-dimensional structure of the acetylcholinesterase-fasciculin complex not only validates the use of sequence comparison for identifying important residues, but in addition reveals unexpected interactions between toxin and target that ase impossible to predict from sequence alone.
1996
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(1996) FEBS Letters. 386, 1, p. 65-71 Abstract
The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter-like back door near Trp84, a key residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the Ω loop, bearing Trp84, with the lid which sequesters the substrate in neutral lipases displaying structural homology with aeetylcholinesterase, suggests a flap-like back door. Both possibilities were examined by site-directed mutagenesis. The shutter-like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap-like back door was tested by de novo insertion of a disulfide bridge which tethered the Ω loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however substantial mobility of the Ω loop in the immediate vicinity of Trp84, even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible.
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(1996) Journal of Research of the National Institute of Standards and Technology. 101, 3, p. 231-241 Abstract
Protein Data Bank (PDB) is an archive of experimentally determined three-dimensional structures of proteins, nucleic acids, and other biological macromolecules with a 25 year history of service to a global community. PDB is being replaced by 3DB, the Three-Dimensional Database of Biomolecular Structures that will continue to operate from Brookhaven National Laboratory. 3DB will be a highly sophisticated knowledge-based system for archiving and accessing structural information that combines the advantages of object oriented and relational database systems. 3DB will operate as a direct-deposition archive that will also accept third-party supplied annotations. Conversion of PDB to 3DB will be evolutionary, providing a high degree of compatibility with existing software.
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(1996) Biophysical Journal. 70, 4, p. 1603-1608 Abstract
Acetylcholinesterase (ACHE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (±10%), which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value for the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus ACHE, which has been constructed from its amine acid sequence, in analogy to the crystal structure of ACHE from Torpedo californica.
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(1996) Protein Science. 5, 4, p. 672-679 Abstract
A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin- mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2- mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 Å resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6122 or P6522 with unit cell dimensions a = b = 75.5 Å, c = 556 Å, giving a V(m) value of 3.1 Å3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.
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(1996) Journal of the American Chemical Society. 118, 10, p. 2340-2346 Abstract
The structure of a complex of Torpedo calfornica acetylcholinesterase with the transition state analog inhibitor m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone has been solved by X-ray crystallographic methods to 2.8 Angstrom resolution. Since the inhibitor binds to the enzyme about 10(10)-fold more tightly than the substrate acetylcholine, this complex provides a visual accounting of the enzyme-ligand interactions that provide the molecular basis for the catalytic power of acetylcholinesterase. The enzyme owes about 8 kcal mol(-1) of the 18 kcal mol(-1) of free energy of stabilization of the acylation transition state to interactions of the quaternary ammonium moiety with three water molecules, with the carboxylate side chain of E199, and with the aromatic side chains of W84 and F330. The carbonyl carbon of the trifluoroketone function interacts covalently with S200 of the S200-H440-E327 catalytic triad. The operation of this triad as a general acid-base catalytic network probably provides 3-5 kcal mol(-1) of the free energy of stabilization of the transition state. The remaining 5-7 kcal mol(-1) of transition state stabilization probably arises from tripartite hydrogen bonding between the incipient oxyanion and the NH functions of G118, G119, and A201. The acetyl ester hydrolytic specificity of the enzyme is revealed by the interaction of the CF3 function of the transition state analog with a concave binding site comprised of the residues G119, W233, F288, F290, and F331. The highly geometrically convergent array of enzyme-ligand interactions visualized in the complex described herein envelopes the acylation transition state and sequesters it from solvent, this being consistent with the location of the active site at the bottom of a deep and narrow gorge.
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(1996) Biochemistry. 35, 33, p. 10854-10861 Abstract
Acetylcholinesterase and butyrylcholinesterase both rapidly hydrolyze the neurotransmitter acetylcholine. The unusual three-dimensional structure of acetylcholinesterase, in which the active site is located at the bottom of a deep and narrow gorge, raises cogent questions concerning traffic of the substrate, acetylcholine, and the products, choline and acetate, to and from the active site. Time-resolved crystallography offers a promising experimental approach to investigate this issue but requires a suitable triggering mechanism to ensure efficient and synchronized initiation of the dynamic process being monitored. Here we characterize the properties of two photolabile triggers which may serve as tools in time-resolved crystallographic studies of the cholinesterases. These compounds are 2- nitrobenzyl derivatives of choline and of carbamylcholine, which generate choline and carbamylcholine, respectively upon photochemical fragmentation. Both photolabile compounds are reversible inhibitors, which bind at the active sites of acetylcholinesterase and butyrylcholinesterase with inhibition constants in the micromolar range, and both photofragmentation processes occur rapidly and with a high quantum yield, without substantial photochemical damage to the enzymes. Photolysis both of acetylcholinesterase and of butyrylcholinesterase, complexed with a 2-nitrobenzyl derivative of choline, resulted in regeneration of enzymic activity. Photolysis of acetylcholinesterase complexed with the 2-nitrobenzyl derivative of carbamylcholine led to time-dependent inactivation, resulting from carbamylation of acetylcholinesterase, which could be reversed upon dilution, due to decarbamylation. Both sets of experiments demonstrated release of choline within the active site. In the former case, choline was produced photochemically at the active site. In the latter case, choline was generated enzymatically, within the active site, concomitantly with carbamylation of the acetylcholinesterase. The two photolabile compounds may thus serve as complementary probes for time-resolved studies of the route of product release from the active sites of the cholinesterases.
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(1996) Nature Structural Biology. 3, 5, p. 452-458 Abstract
Haloarcula marismortui is an archaebacterium that flourishes in the world's saltiest body of water, the Dead Sea. The cytosol of this organism is a supersaturated salt solution in which proteins are soluble and active. The crystal structure of a 2Fe-2S ferredoxin from H. marismortui determined at 1.9 Å is similar to those of plant-type 2Fe-2S ferredoxins of known structure, with two important distinctions. The entire surface of the protein is coated with acidic residues except for the vicinity of the iron-sulphur cluster, and there is an insertion of two amphipathic helices near the N- terminus. These form a separate hyperacidic domain whose postulated function to provide extra surface carboxylates for solvation. These data and the fact that bound surface water molecules have on the average 40% more hydrogen bonds than in a typical non-halophilic protein crystal structure support the notion that haloadaptation involves better water binding capacity.
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(1996) Biopolymers. 38, 1, p. 109-117 Abstract
Multiconfiguration thermodynamic integration was used to determine the relative binding strength of tacrine and 6-chlorotacrine by Torpedo californica acetylcholinesterase. 6-Chlorotacrine appears to be bound stronger by 0.7 ± 0.4 kcal/mol than unsubstituted tacrine when the active site triad residue His-440 is deprotonated. This result is in excellent agreement with experimental inhibition data on electric eel acetylcholinesterase. Electrostatic Poisson-Boltzmann calculations confirm that order of binding strength, resulting in ΔG of binding of-2.9 and -3.3 kcal/mol for tacrine and chlorotacrine, respectively, and suggest inhibitor binding does not occur when His-440 is charged. Our results suggest that electron density redistribution upon tacrine chlorination is mainly responsible for the increased attraction potential between protonated inhibitor molecule and adjacent aromatic groups of Phe-330 and Trp-84.
1995
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(1995) Structure. 3, 12, p. 1355-1366 Abstract
Background: Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. Results The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 å resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (∼2000 å2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. Conclusion Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.
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(1995) FEBS Letters. 371, 3, p. 231-235 Abstract
The primary sequence of esterases from Acinetobacter lwoffii RAG-1 and A. calcoaceticus BD413 were compared with linearized structural sequences of two hundred proteins selected from Brookhaven Protein DataBank using a modified version of the Bowie et al. algorithm [3]. Significant structural homology was found to α/β proteins and specifically to those with the α/β-hydrolase fold for which the crystal structure was reported. No such homology was detected using common primary sequence alignment programs such as FASTA or BLAST.
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(1995) Science. 267, 5202, p. 1344-1346 Abstract
The high-resolution structure of halophilic malate dehydrogenase (hMDH) from the archaebacterium Haloarcula marismortui was determined by x-ray crystallography. Comparison of the three-dimensional structures of hMDH and its nonhalophilic congeners reveals structural features that may promote the stability of hMDH at high salt concentrations. These features include an excess of acidic over basic residues distributed on the enzyme surface and more salt bridges present in hMDH compared with its nonhalophilic counterparts. Other features that contribute to the stabilization of thermophilic lactate dehydrogenase and thermophilic MDH-the incorporation of alanine into or helices and the introduction of negatively charged amino acids near their amino termini, both of which stabilize the α helix as a result of interaction with the positive part of the α-helix dipole - also were observed in hMDH.
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Structures of complexes of acetylcholinesterase with covalently and non-covalently bound inhibitors(1995) Enzymes Of The Cholinesterase Family. p. 59-65 Abstract
Keywords: Biochemistry & Molecular Biology; Cell Biology; Pharmacology & Pharmacy
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Studies on partially unfolded states of Torpedo californica acetylcholinesterase(1995) Enzymes Of The Cholinesterase Family. p. 77-82 Abstract[All authors]
Keywords: Biochemistry & Molecular Biology; Cell Biology; Pharmacology & Pharmacy
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3-D structure of acetylcholinesterase and its complexes with anticholinesterase agents(1995) Modelling Of Biomolecular Structures And Mechanisms. 27, p. 455-460 Abstract
Keywords: ACETYLCHOLINESTERASE; ANTICHOLINESTERASE; AROMATIC RINGS
1994
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(1994) Biochemistry. 33, 48, p. 14407-14418 Abstract
Chemical modification of Torpedo califomica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluoresence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is \u201cnative-like\u201d, whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurialmodified enzyme, is partially unfolded and displays many of the features of the \u201cmolten globule\u201d state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurialmodified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228Oγ and with the main-chain nitrogen and carbonyl oxygen of the same serine residue.
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Open "back door" in a molecular dynamics simulation of acetylcholinesterase(1994) Science. 263, 5151, p. 1276-1278 Abstract
The enzyme acetylcholinesterase generates a strong electrostatic field that can attract the cationic substrate acetylcholine to the active site. However, the long and narrow active site gorge seems inconsistent with the enzyme's high catalytic rate. A molecular dynamics simulation of acetylcholinesterase in water reveals the transient opening of a short channel, large enough to pass a water molecule, through a thin wall of the active site near tryptophan-84. This simulation suggests that substrate, products, or solvent could move through this "back door," in addition to the entrance revealed by the crystallographic structure. Electrostatic calculations show a strong field at the back door, oriented to attract the substrate and the reaction product choline and to repel the other reaction product, acetate. Analysis of the open back door conformation suggests a mutation that could seal the back door and thus test the hypothesis that thermal motion of this enzyme may open multiple routes of access to its active site.
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Differential effects of "peripheral" site ligands on Torpedo and chicken acetylcholinesterase(1994) Molecular Pharmacology. 45, 2, p. 335-340 Abstract
Comparison of the effect of three 'peripheral' site ligands, propidium, d-tubocurarine, and gallamine, on acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) of Torpedo and chicken shows that all three are substantially more effective inhibitors of the Torpedo enzyme than of the chicken enzyme. In contrast, edrophonium, which is directed to the "anionic" subsite of the active site, inhibits the chicken and Torpedo enzymes equally effectively. Two bisquaternary ligands, decamethonium and 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are believed to bridge the anionic subsite of the active site and the "peripheral" anionic site, are much weaker inhibitors of the chicken enzyme than of Torpedo acetylcholinesterase, whereas the shorter bisquaternary ligand hexamethonium inhibits the two enzymes similarly. The concentration dependence of activity towards the natural substrate acetylcholine is almost identical for the two enzymes, whereas substrate inhibition of chicken acetylcholinesterase is somewhat weaker than that of the Torpedo enzyme. The experimental data can be rationalized on the basis of the three-dimensional structure of the Torpedo enzyme and alignment of the chicken and Torpedo sequences; it is suggested that the absence, in the chicken enzyme, of two aromatic residues, Tyr-70 and Trp-279, that contribute to the peripheral site of Torpedo acetylcholinesterase is responsible for the differential effects of peripheral site ligands on the two enzymes.
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(1994) Protein Science. 3, 2, p. 188-197 Abstract
The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 Å from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bisquaternary decamethonium (DECA) ion, acquired during enzyme purification, resides in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.
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STRUCTURE AND DYNAMICS OF PROTEINS - FOREWORD(1994) Israel Journal of Chemistry. 34, 2, p. 149-149 Abstract
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(1994) Biochemical Society Transactions. 22, 3, p. 745-749 Abstract
Keywords: TORPEDO-CALIFORNICA; LIGAND-BINDING; SITE; BUTYRYLCHOLINESTERASE; MECHANISM; DYNAMICS; RECEPTOR; PROTEIN; PROBES; GORGE
1993
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Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase(1993) Proceedings of the National Academy of Sciences of the United States of America. 90, 19, p. 9031-9035 Abstract
Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-Å resolution. In a complex with edrophonium, the quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labeling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.
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(1993) Glycoconjugate Journal. 10, 4, p. 276-277 Abstract
Keywords: Biochemistry & Molecular Biology
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(1993) FEBS Letters. 328, 1-2, p. 165-168 Abstract
The crystal structure of the complex formed between the egg-white biotin-binding protein, avidin, and the dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), was determined to a resolution of 2.5 A. The interaction of avidin with the benzoate ring of HABA is essentially identical to that of the complex formed between HABA and streptavidin (the bacterial analogue of the egg-white protein). This interaction emulates the definitive high-affinity interaction of both proteins with the ureido moiety of biotin. The major difference between the avidin- and streptavidin-HABA complexes lies in their interaction with the hydroxyphenyl ring of the dye molecule; in avidin, two adjacent amino acid residues (Phe72 and Ser73), which are not present in streptavidin, form additional interactions with this ring. These are suggested to account for the higher affinity of avidin for HABA. The characteristic red shift, which accompanies the interaction of both proteins with the dye, was traced to a proposed charge-transfer complex formed between the hydroxyphenyl ring of HABA and the indole ring of Trp70 in avidin (Trp79 in streptavidin). Comparison of binding site residues of two such similar proteins versus their markedly different affinities for two such different substrates should eventually contribute to a better design of biomimetic reagents and drugs.
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(1993) Journal of Computer-Aided Molecular Design. 7, 4, p. 457-472 Abstract
Proteins tend to use recurrent structural motifs on all levels of organization. In this paper we first survey the topics of recurrent motifs on the local secondary structure level and on the global fold level. Then, we focus on the intermediate level which we call the short structural motifs. We were able to identify a set of structural building blocks that are very common in protein structure. We suggest that these building blocks can be used as an important link between the primary sequence and the tertiary structure. In this framework, we present our latest results on the structural variability of the extended strand motifs. We show that extended strands can be divided into three distinct structural classes, each with its own sequence specificity. Other approaches to the study of short structural motifs are reviewed.
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Three-dimensional structures of avidin and the avidin-biotin complex(1993) Proceedings of the National Academy of Sciences of the United States of America. 90, 11, p. 5076-5080 Abstract
The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel β-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.
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(1993) Current Opinion in Structural Biology. 3, 3, p. 323-335 Abstract
During the past year, X-ray crystallographic studies of DNA have begun to focus not only on the relationship between base sequence and the fine structure of DNA, but more on unusual structures such as quadraplexes and bulges. In the study of drug-DNA complexes, more attention is being directed toward rational drug design.
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(1993) Chemico-Biological Interactions. 87, 1-3, p. 187-197 Abstract
Based on our recent X-ray crystallographic determination of the structure of acetylcholinesterase (AChE) from Torpedo californica, we can see for the first time, at atomic resolution, a protein binding pocket for the neurotransmitter, acetylcholine. It was found that the active site consists of a catalytic triad (S200-H440-E327) which lies close to the bottom of a deep and narrow gorge, which is lined with the rings of 14 aromatic amino acid residues. Despite the complexity of this array of aromatic rings, we suggested, on the basis of modelling which involved docking of the acetylcholine (ACh) molecule in an all-trans configuration, that the quaternary group of the choline moiety makes close contact with the indole ring of W84. In order to study the interaction of AChE with anticholinesterase drugs at the structural level, we have incorporated into the acetylcholinesterase crystals several different inhibitors, and have recently determined the 3-D structure of AChE:edrophonium and AChE:tacrine complexes. The crystal structures of both of these complexes are in good agreement with our model building of the ACh bound in the active site of AChE and indicate the interactions of these two drugs with the enzyme.
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(1993) Proceedings of the National Academy of Sciences of the United States of America. 90, 11, p. 5128-5132 Abstract
Electrostatic calculations based on the recently solved crystal structure of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) indicate that this enzyme has a strong electrostatic dipole. The dipole is aligned with the gorge leading to its active site, so that a positively charged substrate will be drawn to the active site by its electrostatic field. Within the gorge, aromatic side chains appear to shield the substrate from direct interaction with most of the negatively charged residues that give rise to the dipole. The affinity of quaternary ammonium compounds for aromatic rings, coupled with this electrostatic force, may work in concert to create a selective and efficient substrate-binding site in acetylcholinesterase and explain why the active site is situated at the bottom of a deep gorge lined with aromatic residues.
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(1993) Protein Science. 2, 5, p. 814-825 Abstract
The threedimensional Xray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P21212 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787788). Refinement of the protein structure (3, 148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an Rfactor of 0.207 using all data from 8.0 to 3.2 Å resolution and group thermal parameters. The monomermonomer interactions typical of bacterial Fe and Mncontaining SODs are retained in the human enzyme, but the dimerdimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84° relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimerdimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metalligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.
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(1993) Protein Science. 2, 3, p. 366-382 Abstract
Based on the recently determined Xray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their threedimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two Xray structures.
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(1993) Biochemistry. 32, 8, p. 2116-2119 Abstract
Alternating purine-pyrimidine DNA sequences such as poly[d(C-G)] or poly[d(m5C-G)] undergo a cooperative, salt-induced structural transition from a right-handed B conformation, which prevails at relatively low ionic strength, into a left-handed Z form, generally believed to be stabilized by high salt concentrations. We report here that upon a monotonous increase of the ionic strength, the well-established B to Z transition is followed by a second, hitherto unobserved conformational change leading from Z-DNA back into a right-handed B-like form. This observation indicates that, in contrast with the current convention, the Z motif represents an unstable configuration relative to the B form at both low and high salt concentrations and that the occurrence of a left-handed DNA structure, presently depicted as a step function of the ionic strength, should rather be treated in terms of a pulse. The reported transition underscores the inherent metastability of the Z configuration, and indicates, consequently, that this motif is ideally suited to act as a structural regulatory element, as such an element should be endowed with a large susceptibility to cellular parameters.
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(1993) Biochemistry. 32, 2, p. 401-403 Abstract
Brownian dynamics simulations have been used to calculate the diffusion-controlled rate constants for the binding of a positively charged ligand to several models of acetylcholinesterase (AChE). The crystal structure was used to define the detailed topography and the active sites of the dimeric enzyme. The electric field around AChE was then computed by solving the Poisson equation for different charge distributions in the enzyme at zero ionic strength. These fields were used in turn to calculate the forces on the diffusing ligand. Significant increases in the rate constant resulted in going from a model with no charges to one with the net charges concentrated at the centers of the monomers and then to a model with a realistic distribution of charges throughout the enzyme. The results show that electrostatic steering of ligands contributes to the high rate constants that are observed experimentally for AChE.
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(1993) Progress in Brain Research. 98, C, p. 139-146 Abstract
Vertebrates possess two cholinesterases, acetylcholinesterase and butyrylcholinesterase. Cholinesterases catalyze a very simple reaction, hydrolysis of the ester bond of acetylcholine. The role of AChE in cholinergic transmission, although admittedly secondary to that of the pre-synaptic machinery responsible for the synthesis and release of acetylcholine, and of the postsynaptic receptors, is crucial for synaptic function. The role of ChE is not clear; this enzyme is, in fact, dispensable, since its absence in humans does not correlate with any physiological abnormality. Despite the fact that they do not, at first sight, appear as glamorous as receptors, cholinesterases display a number of fascinating features, and pose important questions concerning their structure and functions. AChE is one of the fastest enzymes known and possesses an unusual molecular structure. Both AChE and BChE display a repertoire of molecular forms, which differ in their quaternary structure and may be anchored in different ways to synaptic structures. Cholinesterases are thought to exert non-cholinergic functions, e.g. in morphogenesis, during early embryonic development, in the modulation of neuronal activity and in the elimination of various toxic compounds, which may explain their presence outside the context of cholinergic transmission. They are also expressed abnormally in some tumors and in other pathological states. This chapter discusses the following aspects: the atomic structure of cholinesterases and their catalytic mechanism; the structure and biosynthesis of their molecular forms; the possibility that these enzymes participate in cellular interactions in addition to their catalytic activity.
1992
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(1992) Journal of biomolecular structure & dynamics. 10, 2, p. 317-331 Abstract
Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5 pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5 Py Pu 3, a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5 pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5 pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.
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(1992) FEBS Letters. 309, 3, p. 421-423 Abstract
Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterass. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326 → Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gin or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.
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Mutagenesis of human acetylcholinesterase: Identification of residues involved in catalytic activity and in polypeptide folding(1992) Journal of Biological Chemistry. 267, 25, p. 17640-17648 Abstract[All authors]
Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
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(1992) Journal of Molecular Biology. 225, 2, p. 397-431 Abstract
The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 Å resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A · A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 Å resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.
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(1992) Protein Engineering, Design and Selection. 5, 3, p. 197-211 Abstract[All authors]
We have identified a new protein fold-the α/β hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an α/β sheet, not barrel, of eight β-sheets connected by α-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the α/β hydrolase fold enzymes.
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(1992) Proceedings of the National Academy of Sciences of the United States of America. 89, 22, p. 10827-10831 Abstract
Torpedo acetylcholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholinesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature of the AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEase-selective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the activesite gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.
1991
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(1991) Journal of Molecular Biology. 221, 3, p. 909-918 Abstract
"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of γ-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-γ-chymotrypsin conjugates were obtained by prolonged soaking of native γ-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-γ-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-γ-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 Oγ. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2·6 Å) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 Nε2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3·6 Å away from His57 Nε2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.
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(1991) Biochemistry. 30, 21, p. 5217-5225 Abstract
The determination of three separate γ-chymotrypsin structures at different temperatures and resolutions confirmed the presence of electron density in the active site, which could be interpreted as an oligopeptide as had previously been suggested by Dixon and Matthews [(1989) Biochemistry 28, 70337038]. HPLC analyses of the enzyme before and after crystallization demonstrated the presence of a wide variety of oligopeptides in the redissolved crystal, most with COOH-terminal aromatic residues, as expected of the products of chymotrypsin cleavage, which appeared to arise from extensive autolysis of the enzyme under the crystallization conditions. The refined structures agree well with the conformation of both γ-chymotrypsin and a-chymotrypsin. The electron density in the active site is thus interpreted as arising from a repertoire of autolysed oligopeptides produced concomitantly with crystallization. The COOH-terminal carbons of the polypeptide(s) display short contact distances (1.97, 2.47, and 2.13 Å, respectively) to Serl95 Oγ in all three refined structures, but the electron density is not continuous between these two atoms in any of them. This suggests that some sequences are covalently bound as enzyme intermediates while others are noncovalently bound as enzyme-product complexes.
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(1991) Science. 253, 5022, p. 872-879 Abstract
The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol- specific phospholipase C. The enzyme monomer is an α/β protein that contains 537 amino acids. It consists of a 12-stranded mixed β sheet surrounded by 14 α helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.
1990
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(1990) Biopolymers. 30, 5-6, p. 499-508 Abstract
It is possible to construct fragments of protein structures by using the known values for the fixed bond lengths, bond angles, and torsion angles, and \u201cdialing\u201d in the dihedral angles ϕ and ψ. By choosing these angles in different ways, it is possible to create different populations of fragments and to investigate their properties. We analyzed the following populations: Real fragments taken randomly from known structures. Reconstructed fragments, which are constructed, using the \u201cfixed geometry\u201d assumption, from a set of consecutive pairs of dihedral angles drawn from known structures. Random fragments that are constructed from a random set of dihedral angles from known structures, and doubletpreserving fragments, which are constructed from a set of dihedral angles drawn at random from known structures in a way such that the distribution of two consecutive pairs of dihedral angles in this population is similar to that distribution in the known structures. We examine the fixed geometry assumption and demonstrate that even reconstructed fragments contain many atomic collisions. We show that random fragments have only slightly more interatomic collisions than the reconstructed fragments. Nevertheless, the population of random fragments is structurally different from the population of reconstructed fragments. On the other hand, we show that the doubletpreserving fragments exhibit properties that are similar to the real population. Thus the doublet preserving random population can be used to simulate the structure of short polypeptides.
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(1990) Methods in enzymology. 184, C, p. 90-93 Abstract
This chapter describes various types of crystal forms of Avidin. The first single crystal forms of egg-white Avidin were obtained using the dialysis method at room temperature by equilibrating a solution containing 1020 mg/ml of protein versus a sodium phosphate buffer at pH 5.2. Crystals appeared when the buffer concentration was increased to about 3.0 M. A different crystal form was obtained from polyethylene glycol (PEG) 6000. These crystals were grown using the batch method 3 at room temperature. The protein solution contained 2045 mg/ml protein, 10 mM sodium phosphate (pH 6.0), and 150 mM NaCI. The carbohydrate moiety of the avidin is not essential for its biotin-binding activity. The biotin-binding properties of the nonglycosylated avidin are equivalent to those obtained for the native (glycosylated) avidin molecule. The method consists of first coating the crystal with viscous oil in the crystallization droplet and removing all traces of mother liquor. The crystal is then picked up with a thin glass spatula, putting it directly under a stream of boiled liquid N2 at a temperature of approximately 90 K.
1989
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(1989) Journal of Molecular Biology. 206, 4, p. 787-788 Abstract
The genetically engineered human manganese superoxide dismutase crystallizes in space group P21212 with a = 75·51 A ̊, b = 79·00 A ̊, c = 67·95 A ̊. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 Å resolution at this temperature.
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(1989) Proteins-Structure Function And Bioinformatics. 5, 4, p. 355-373 Abstract
A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of Cα coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set ofstandard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can \u201creplace\u201d about 76% ofall hexamers in wellrefined known proteins with an error of less than 1 Å, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.
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Protein adaptation to extreme salinity: the crystal structure of 2Fe-2S ferredoxin from Halobacterium marismortui.(1989) Progress in Clinical and Biological Research. 289, p. 171-187 Abstract
1988
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(1988) Journal of Molecular Biology. 203, 3, p. 821-823 Abstract
A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2·0 Å (1 Å = 0·1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163·4(±0·2) A ̊, b = 112·1(±0·2) A ̊, c = 81·3(±0·1) A ̊.
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(1988) Journal of Molecular Biology. 200, 3, p. 609-610 Abstract
Malate dehydrogenase from the extreme halophile Halobacterium marismortui crystallizes in highly concentrated phosphate solution in space group I2 with cell dimensions a = 113.8 A ̊, b = 122.8 A ̊, c = 126.7 A ̊, β = 98.1 °. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell.
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(1988) Journal of biomolecular structure & dynamics. 5, 5, p. 965-979 Abstract
Structural modelling techniques using energy minimization and molecular dynamics have been employed to generate kinked models for the solution structure of two DNA tridecamer sequences containing inserted adenosines: d(CGCAGAATTCGCG)2 and d(CGCAGAGCTCGCG)2. These models are consistent with NMR studies of these sequences in solution. The overall shapes of the two models are similar, consisting of three B-DNA sections: two outer segments on die same side of the central portion, with the additional adenosines acting as wedges to kink the structure. An alternative scheme for the hydrogen bond pairing at the kink site is suggested as a way for the additional adenosines to be stabilized in the duplex.
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(1988) Nature. 334, 6177, p. 82-84 Abstract
Unpaired bases in DNA have been assigned a possible role in the mechanism of frameshift mutagenesis in sequences with repeated base pairs1. They also occur in quasipalindromic DNA sequences, which have been implicated in mutagenesis where there are no repeated base pairs, through the formation of single-stranded hairpin loops2,3. The conformation of unpaired bases in DNA has been the subject of numerous thermodynamic as well as high resolution NMR (nuclear magnetic resonance) studies (reviewed in ref. 4). The NMR studies in solution5 have shown that the duplex of the tridecamer DNA fragment d(CGCAGAATTCGCG) remains intact, and that the unpaired adenosines are stacked into the duplex. Having crystallized this oligonucleotide and determined its structure, we find its conformation in the crystal is close to that of a B-DNA duplex, with the two additional adenosines looped out from the double helix and causing little disruption of the rest of the structure.
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(1988) Nature. 334, 6177, p. 85-86 Abstract
Errors during DNA replication or repair can lead to the presence of unpaired or inserted bases in the double helix, as well as to mismatched base pairs. So far only structures of the latter type have been characterized by X-ray crystallography. We report here a 3-Å crystal structure of DNA 15-mer d(CGCGAAATTTACGCG), which forms a duplex with two unpaired adenine residues looped outside the B-type helix. This arrangement is in disagreement with the nuclear magnetic resonance spectroscopy results for the same 15-mer in solution, indicating polymorphic nature of the structure adopted by this sequence.
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A kinked model of a DNA tridecamer with an unpaired adenosine: energy minimization and X-ray structural studies(1988) in ''Molecular Structure: Chemical Reactivity and Biological Activity". p. 195-200 [All authors]
1987
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(1987) The Biochemical journal. 247, 2, p. 277-285 Abstract
The nucleotide sequence was determined for part of the Klebsiella pneumoniae nif gene cluster containing the 3' end of the nifD gene and the entire length of the nifK gene (encoding the alpha- and beta-subunits of the nitrogenase MoFe protein respectively), as well as the putative start of the nifY gene, a gene of as yet unknown function. A broad-based comparison of a number of MoFe protein alpha-subunits, beta-subunits and alpha-versus beta-subunits was carried out by the use of a computer program that simultaneously aligns three protein sequences according to the mutation data matrix of Dayhoff. A new kind of quantitative statistical measure of the similarity between the aligned sequences was obtained by calculating and plotting standardized similarity scores for overlapping segments along the aligned proteins. This calculation determines if a test sequence is similar to the consensus sequence of two other proteins that are known to be related to each other. The different beta-subunits compared were found to be significantly similar along most of their sequence, with the exception of two relatively short regions centred around residues 225 and 300, which contain insertions/deletions. The overall pattern of similarity between different alpha-subunits exhibits resemblance to the overall pattern of similarity between different beta-subunits, including regions of low similarity centred around residues 225 and 340. Comparison of alpha-subunits with beta-subunits showed that a region of significant similarity between the two types of subunits was located approximately between residues 120 and 180 in both subunits, but other parts of the proteins were only marginally similar. These results provide insights into likely tertiary structural features of the MoFe protein subunits
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(1987) Nucleic Acids Research. 15, 9, p. 3877-3890 Abstract
Conformational transitions for a series of imperfect palindromes related to the dodecamer d(CGCGAATTCGCG) have been investigated. These sequences are: two isomeric 13-mers - d(CGCGAATTCGCG) (13-merI) and d(CGCGAATTACGCG) (13-merII), 17-mer d(CGCGCGAATTACGCGCG) and 15-mer d(CGCGAAATTTACGCG). Insertion of a single adenine nucleotide prevents these sequences from being self-complementary. Analysis of thermodynamic parameters derived from the melting profiles together with other data at higher concentrations (NMR and calorimetry) indicates that the insertion of the additional nucleotide which lacks a complement in the opposite strand does not change the enthalpy of the duplex formation, but does alter the number of stable nucleation configurations. The relative position of the insertion within the self-complementary sequence determines the equilibrium between the duplex form and the single-stranded hairpin loop. C-G segments separated by the insertion from the rest of the molecule can undergo an independent conformational transition at high salt concentration, probably to the Z form.
1986
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(1986) Bioinformatics. 2, 4, p. 283-289 Abstract
We present a fast algorithm to produce a graphic matrix representation of sequence homology. The algorithm is based on lexicographical ordering of fragments. It preserves most of the options of a simple naive algorithm with a significant increase in speed. This algorithm was the basis for a program, called DNAMAT, that has been extensively tested during the last three years at the Weizmann Institute of Science and has proven to be very useful. In addition we suggest a way to extend our approach to analyse a series of related DNA or RNA sequences, in order to determine certain common structural features. The analysis is done by 'summing' a set of dot-matrices to produce an overall matrix that displays structural elements common to most of the sequences. We give an example of this procedure by analysing tRNA sequences.
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(1986) Biochemistry. 25, 23, p. 7417-7423 Abstract
We have studied the tridecadeoxynucleotide CGCGAATTACGCG (I), which contains an additional A at position 9 compared to the dodecanucleotide of which the crystal structure has been determined. Sequence I exhibits no distinct melting curve and also has a concentration-dependent pattern of peaks on reverse-phase chromatography. This behavior is explained by a slow equilibration between loop and duplex forms in solution. We have characterized this equilibrium by proton NMR spectroscopy and shown that it is fully reversible by monitoring the two thymine methyl resonances, each of which occurs in two environments. Lower temperature and higher concentration favor the duplex; the midpoint of the transition is such that the loop predominates at room temperature. We have measured the van't Hoff enthalpy of formation of the duplex and the activation energy by temperature-jump and saturation-transfer experiments. The results are compared with those for the 17-mer sequence CGCGCGAATTACGCGCG (II), which contains two additional base pairs in the stem of the loop. The thermodynamic parameters and the effect of increasing salt concentration on the rate of conversion of the loop and duplex forms lead us to presume that the mechanism of interconversion involves complete strand separation and re-formation rather than cruciform formation and branch migration.
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(1986) FEMS Microbiology Letters. 39, 1-2, p. 129-135 Abstract
Three proteins from Halobacterium marismortui, malate dehydrogenase (hMDH), glutamate dehydrogenase (hGDH) and ferredoxin (hFD) were purified and characterized with respect to their molecular masses, amino acid composition and, for hFD only, primary structure. Striking features of halophilic proteins are: the high excess of acidic over basic residues; acidic clusters in the sequence. Low-salt concentration causes inactivation and changes in structural parameters of hMDH and hGDH. Reactivation of hMDH involves long-lived stable intermediates. The salt concentration optimum of enzymic activity is independent of salt nature. The high capacity of halophilic proteins to retain water and salt is due to unique molecular properties, studied by physico-chemical techniques.
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(1986) The EMBO Journal. 5, 7, p. 1719-1725 Abstract
Structural modelling techniques are employed to explore the energetic requirements for the transformation of classical B DNA into unwound yet doublestranded DNA structures. Structural idealization using CORELS computer program of Sussman et al. followed by energy minimization using the EREF program of Levitt, leads to two regular nonhelical models. In both models, the bases are conventionally paired and stacked, yet there is no net rotation between successive base pairs. One model, N1, has a 1bp repeating unit; the second, N2, has a 2bp repeating unit. The dihedral angles of the backbone all have values found either in the B or the Z form of DNA, except for the PO5C5C4 angle, which is in the unprecedented g+ or g domains. The energy difference found between the two N form models and B form DNA are 6.6 and 3.4 kcal/mol/nucleotide for N1 and N2 respectively. These relatively low energy differences encourage the idea that nonhelical forms of DNA may contribute to the alternate DNA structures found in S1 nuclease sensitive and other regulatory regions of active genes.
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(1986) Trends in Biochemical Sciences. 11, 4, p. 155-156 Abstract
An interpretation of the particular proportions of DNA as found in the Watson-Crick double helix model is suggested. It is based on the classical, highly aesthetical concept known as the Golden Ratio. Specifically, it is shown that in B-DNA both pitch/diameter and diameter/offset are extremely close to the Golden Ratio. Here, pitch is the helical repeat, and offset refers to the vertical distance that forms the minor groove.
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(1986) Journal of Molecular Biology. 188, 1, p. 111-113 Abstract
Crystals of the DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G) have been grown by the vapor-diffusion technique with 2-methyl-2,4-pentanediol as precipitant. They are monoclinic space group (C2, with a = 79.6 A ̊, b = 43.1 A ̊, c = 24.9 A ̊ and β = 98.7 °. Previous nuclear magnetic resonance studies predicted that this tridecamer forms a duplex similar to the B DNA dodecamer, d(C-G-C-G-A-A-T-T-C-G-C-G), except for an extra adenosine residue that is stacked within the helix but remains unpaired: {A figure is presented} Preliminary X-ray diffraction studies confirmed that the tridecamer is in the B DNA conformation, consistent with the nuclear magnetic resonance results.
1985
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(1985) Methods in enzymology. 115, C, p. 271-303 Abstract
The CORELS (COnstrained-REstrained Least-Squares) program is developed for proteins and nucleic acids to take advantage of the intrinsic rigid groups found in the molecules and to overcome the relatively low resolution of the X-ray data from their crystals. CORELS combines Scheringer's rigid groups constraints, extended to allow for variable torsion angles, with distance restraints to maintain stereochemistry between groups within a specified error limit. Even though allowing variable internal dihedral angles introduces torsional degrees of freedom within the otherwise constrained group, it can reduce the total number of structural parameters in the structure by decreasing the number of groups. The advantages of this approach include a large increase in the data-to-parameter ratio over the restrained refinement methods; automatic maintenance of group stereochemistry: within the group, all bond lengths and bond angles are constrained. The use of a constrained-restrained least-squares procedure has proven to be extremely useful in refining macromolecular structures, especially when the initial model has severe errors. This method inherently has many fewer degrees of freedom than restrained refinement procedures and therefore is applicable at extremely low resolution with a very large radius of convergence.
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(1985) Proceedings of the National Academy of Sciences of the United States of America. 82, 10, p. 3073-3077 Abstract
Currently there are several computer algorithms available for aligning two biological sequences. When more than two sequences are to be aligned, however, pairwise comparisons using these methods rarely lead to a consistent alignment of the sequences. One obvious solution to this problem is to compare all the sequences simultaneously. Here we present an algorithm for the simultaneous comparison of three biological sequences. The algorithm is an extension of the method developed by S.B. Needleman and C.D. Wunsch, but it decreases the almost prohibitively long computing time required by a direct naive extension to a practical level: it takes time proportional to the cube of the mean sequence length, in comparison to the fifth power time taken by the direct extension. Simultaneous comparison not only gives a consistent alignment of the three sequences, but it could also reveal homologous residues in the sequences that might be overlooked by the pairwise comparisons. As an example of the application of the algorithm, three copper-containing proteins, plastocyanin, stellacyanin, and cucumber basic blue protein, are compared.
1983
1982
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(1982) Nucleic Acids Research. 10, 8, p. 2701-2708 Abstract
Analyses of secondary structures proposed for ribosomal RNA's show that, of the different kinds of base pairs directly adjoining the ends of postulated double-helical regions, only A-G with A at the 5' end significantly exceeds the number expected for a random base distribution. An A(syn)-G(trans) hydrogen-bonded basepair is proposed. This could fit at the end of an undistorted double helix, but would prevent further base stacking, thus favoring a break in the double helix to produce a non-linear tertiary structure.
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(1982) Journal of the American Chemical Society. 104, 1, p. 270-278 Abstract
A series of statistical computations have been carried out to test various theoretical potential energy estimates of furanose pseudorotation. Energy surfaces describing the flexibility of both ribose and deoxyribose have been compared through a Boltzmann analysis with each other and also with published X-ray and NMR measurements of pseudorotation (i.e., with distributions of solid state puckerings, standard valence angle geometries, and mean vicinal coupling constants in RNA and DNA analogues). No single theoretical approach is able to account simultaneously for the experimental properties of both ribose and deoxyribose. Methods generally satisfactory for ribose pseudorotation are unsuitable for deoxyribose and vice versa. In the commonly occurring mononucleosides and -nucleotides the pseudorotational motions are decidedly \u201cstif\u201d with the potential energy barrier somewhat higher for ribose than for deoxyribose. When incorporated into a polynucleotide backbone, this local stiffness is a major determinant of chain flexibility and a characteristic difference between RNA and DNA systems.
1981
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(1981) Gene. 13, 4, p. 355-364 Abstract
We present a method which permits comparison of the preferential use of degenerate codons within any gene. The method makes use of the triplet frequencies in the noncoding frames to assess whether a preference is specific to the reading frame. Preference is given a statistical meaning by use of the analysis of variance coupled to Duncan's multiple range test. Preferential use of degenerate codons is gene-specific and independent of gene size. The data suggest that any correlation between codon frequency distribution and tRNA levels is unreliable. In those animal genes examined, codons ending in C or G are preferred; in animal viruses tested, codons ending in U or A are preferred. Similarly, the bacterial genes and the genes of single-stranded DNA phages that we analyzed differed from each other as well as from eukaryotic genes in the third base of the codon.
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(1981) Science. 212, 4500, p. 1275-1277 Abstract
A survey of all available double-stranded RNA crystal structures shows that there is a considerable range of variation in local conformation of a given base-pair doublet, but that there is no significant correlation between base-pair sequence and RNA local conformation.
1980
1979
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(1979) Journal of Molecular Biology. 134, 2, p. 375-377 Abstract
The 2 Fe-ferredoxin from the Halobacterium of the Dead Sea has been crystallized. The space group is P6322 with one protein molecule per asymmetric unit. The cell parameters are a = b = 60.6 A ̊, c = 127.8 A ̊. The crystals are stable under radiation and diffract to high resolution.
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(1979) Journal of Molecular Biology. 131, 2, p. 137-155 Abstract
We have determined the crystal structure of demetallized concanavalin A, at a resolution of 3.2 Å, by molecular replacement using the known structure of native concanavalin A. Refinement of the initial model using a constraint-restraint reciprocal-space least-squares procedure caused the conventional crystallographic agreement (R) factor to decrease from 0.47 to a final value of 0.26. There are significant conformational changes in the metal-binding region involving residues Asp 19 and His24, which are substantially closer to each other than in native concanavalin A. These residues form an internal salt bridge which does not exist when the metal ions are attached to the protein. The binding site for transitionmetal ions is still intact, but the calcium site is not, since one of its two carboxylic ligands, Asp 19, is unavailable. Flexibility is observed for one of the transitionmetal ligands, Glu8, as well as for some segments of the backbone. The latter could account for the increased susceptibility of demetallizcd concanavalin A to proteolysis.
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(1979) Nature. 278, 5700, p. 188-190 Abstract
Our understanding of the molecular structure-function relationship in tRNA rests mainly on three types of information. First, on the common sequence patterns which have emerged from careful examination of many primary structures1-3 ; second, a wide variety of spectral and other physical and chemical results must be accounted for by the molecular structure 4 -; and third, there is the detailed image of the yeast tRNAPhe molecule independently determined and refined from two different-albeit similar-crystal forms 7-10 It is also clear, however, that the molecular model deduced from the yeast tRNAPhe crystal structure cannot be easily reconciled with all structural requirements for function and is best considered a well-defined and stable canonical form of tRNA which is packed in an unusually well-ordered way in specific crystal lattices. Notwithstanding the enormous value of this canonical form in explaining the basic architectural features of tRNA, it is clearly important to image other crystalline tRNAs; particularly tRNAs that exhibit different functions (such as, initiators) or have significantly different covalent structures (for example, class III tRNAs) 1 or those that crystallise in different solvent conditions. We report here the initial results of the crystal structure determination of a eukaryotic initiator tRNA crystallised from a highly polar aqueous solvent 11 ' 12 Its architecture is essentially the same as crystalline yeast tRNAPhc, except for a small but significant difference in the position of the anticodon arm.
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Smooth bending of DNA in chromatin(1979) In M. Balaban (Ed.) Molecular mechanisms of biological recognition (Elsevier/North-Holland: Biomedical Press).. p. 227-232
1978
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(1978) Journal of Molecular Biology. 123, 4, p. 607-630 Abstract
We present the results of the final stage of the X-ray Crystallographic studies of yeast phenylalanine transfer RNA in an orthorhombic crystal form. The crystal structure of the transfer RNA has been refined by a least-squares procedure to minimize the difference between the observed (Fo) and calculated (Fc) structure factors from X-ray diffraction patterns. The final Crystallographic discrepancy index, R = ∑|Fo-Fc|/∑Fo, is 0.198, based upon 8426 structure factors with magnitudes over twice the estimated standard deviation, corresponding to 96.4% of the complete set of data with resolutions up to 2.7 Å. During the refinement, bond lengths and angles within each phosphate group and each nucleoside (base plus sugar) were constrained exactly to their appropriate standard values, while those for the linkages between the nucleosides and phosphates were elastically restrained close to their standard values. The details of the application of the constraint-restraint least-squares (CORELS) refinement method to the crystal structure of yeast phenylalanine tRNA are described in this paper. A complete list of atomic co-ordinates and the rigid group thermal factors are presented. The stereochemical details of this structure and their functional implications are described in the following paper.
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(1978) Journal of Molecular Biology. 123, 4, p. 631-660 Abstract
The structural features of yeast phenylalanine transfer RNA are analyzed and documented in detail, based on atomic co-ordinates obtained from an extensive crystallographic refinement of the crystal structure of the molecule at 2.7 Å resolution (see preceding paper). We describe here: the relative orientation and the helicity of the base-paired stems; more definitive assignments of tertiary hydrogen bonds involving bases, riboses and phosphates; binding sites for magnesium hydrates, spermine and water; iriter-molecular contacts and base-stacking; flexibility of the molecule; conformational analysis of nucleotides in the structure. Among the more noteworthy features are a considerable irregularity in the helicity of the base-paired stems, a greater flexibility in the anticodon and aminoacyl acceptor arms, and a "coupling" among several conformational angles. The functional implications of these structural features are also discussed.
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(1978) Proceedings of the National Academy of Sciences of the United States of America. 75, 1, p. 103-107 Abstract
The idea that DNA in chromatin can be packed smoothly, without breaking of base-stacking interactions, has been explored by both energetical estimations and stereochemical model building. A model of deformed DNA is built that fits reasonably to the known dimensions of a nucleosome. The model has slightly changed torsion angles in the sugar-phosphate moieties relative to B DNA, varying gradually along the chains. The angle between planes of adjacent base pairs varies between 1 and 7°. This model of deformed DNA does not have any unusually close nonbonded contacts and is evidently not the only possible model of smooth packing of DNA in chromatin. An energetical estimation of the critical radius of curvature of a smoothly bent DNA molecule is made using approximate potential functions for different van der Waals contacts in the B DNA structure. The critical radius of curvature of the deformed DNA-axis is close to the radius of a nucleosome (~50 Å). The smooth packing is a good alternative to models of kinked folding of DNA in nucleosomes.
1977
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(1977) Nucleic Acids Research. 4, 8, p. 2811-2820 Abstract
X-ray crystallographic studies indicate that there are at least four site-specifically bound hydrated Mg 2+ ions, [Mg(H 2 O) n ] 2+ , in yeast tRNA Phe . The size and the octahedral coordination geometry, rather than the charge, of [Mg(H 2 O) n ] 2+ appear to be the primary reasons for the specificity of magnesium ions in site-binding and in the stabilization of the tertiary structure of tRNA.
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(1977) Proceedings of the National Academy of Sciences of the United States of America. 74, 4, p. 1458-1462 Abstract
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Transfer RNA: structure-function correlation.(1977) Horizons in biochemistry and biophysics. 4, p. 159-200 Abstract
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(1977) Acta Crystallographica Section A. A33, p. 800-804
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Transfer RNA: structure-function correlation(1977) In Quagliariello, (Ed.) Horizons in Biochemistry and Biophysics, (Addison-Wesley Publ. Co., Reading, MA).. p. 159-199
1976
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(1976) Nature. 260, 5552, p. 645-646 Abstract
A PATTERN has been found in the structure of transfer RNA by which a polynucleotide chain can make an abrupt change in direction so as to make a sharp cornered loop or a sharp bend.
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(1976) Science. 192, 4242, p. 853-858 Abstract
Analysis of three sets of atomic coordinates of yeast phenylalanine tRNA establishes common features.
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(1976) Biochemical and Biophysical Research Communications. 68, 1, p. 89-96 Abstract
The atomic coordinates are given for yeast phenylalanine transfer RNA in the orthorhombic crystal form. The structure has been refined by fitting to successively improved electron density maps at 2.7 Å resolution. The model fitting has been accomplished by using an interactive computer graphics system to minimize the errors inherent in manual model building and coordinate measurements, using an optical comparator. The atomic coordinates have then been "idealized" to make bond distances, bond angles, steric conformation and non-bonded contacts close to standard values, while constraining the model to fit the electron density maps.
1975
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(1975) Proceedings of the National Academy of Sciences of the United States of America. 72, 12, p. 4866-4870 Abstract
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The tertiary structure of yeast phenylalanine transfer RNA(1975) In M. Sundaralingam, & S. T. Rao (Eds.), Structure and conformations of nucleic acids and protein-nucleic acid interactions. Baltimore: University Park Press.. p. 7-23 [All authors]
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A model for a recognition scheme between double stranded DNA and proteins(1975) In M. Sundaralingam, & S. T. Rao (Eds.),Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interaction. (Baltimore: University Park Press. .. p. 571-575
1974
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(1974) Proceedings of the National Academy of Sciences of the United States of America. 71, 12, p. 4970-4974 Abstract
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(1974) Science. 185, 4149, p. 435-440 Abstract
The 3-angstrom electron density map of crystalline yeast phenylalanine transfer RNA has provided us with a complete three-dimensional model which defines the positions of all of the nucleotide residues in the molecule. The overall features of the molecule are virtually the same as those seen at a resolution of 4 angstroms except that many additional details of tertiary structure are now visualized. Ten types of hydrogen bonding are identified which define the specificity of tertiary interactions. The molecule is also stabilized by considerable stacking of the planar purities and pyrimidines. This tertiary structure explains, in a simple and direct fashion, chemical modification studies of transfer RNA. Since most of the tertiary interactions involve nucleotides which are common to all transfer RNA's, it is likely that this three-dimensional structure provides a basic pattern of folding which may help to clarify the three-dimensional structure of all transfer RNA's.
1973
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(1973) BBA Section Nucleic Acids And Protein Synthesis. 308, 2, p. 189-197 Abstract
The separation of 2-5 dinucleoside monophosphates from their 3-5 isomers on Sephadex LH-20 column is described. In addition, the CD spectra of all the different dinucleoside monophosphates are given. The correlation of the differences in conformation between the 2-5 and the 3-5 dinucleoside monophosphates with their behaviour on the Sephadex LH-20 column is discussed. It is shown that nucleotides do not behave on the Sephadex LH-20 column according to the rules of gel filtration and it is suggested that the separation of the different dinucleoside monophosphates is dictated by the conformation, which affects the adsorption of the nucleotidic material to the column.
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(1973) Journal of Chromatography A. 79, C, p. 139-146 Abstract
The separation of 2-5 dinucleoside monophosphates from their corresponding 3-5 isomers on QAE-Sephadex A-25 (bicarbonate form) is described. The column is loaded with a mixture of the two isomers and eluted with a linear gradient of ammonium bicarbonate (from 0.02 to 0.2 M) The two isomers appear in two different peaks, and the nucleotidic material is isolated by lyophilization. In addition, the separation of 2-5 dinucleoside monophosphates from the corresponding 3-5 isomers by paper chromatography and by thin-layer chromatography on aluminium cards coated with cellulose powder is described.
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(1973) Acta Crystallographica Section B. B29, p. 2918-2926
1972
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(1972) Journal of Molecular Biology. 66, 3, p. 403-421 Abstract
The crystal structure of uridylyl 3,5-adenosine phosphate (UpA) has been determined and refined to an R value of 0.062. There are two UpA molecules per asymmetric unit in a monoclinic unit cell with dimensions a = 16.91, b = 12.37, c = 11.25 A ̊ and β = 96.0 °. The conformation of the phosphodiester linkage is different for the two molecules; one UpA suggests a model for a sharp turn in a single-stranded nucleic acid structure and the other for a helical structure. All the nucleoside units have similar conformations. There is no complementary base pairing between the adenines and uracils but rather layers of base-paired adenines and base-paired uracils separated by 3.4 Å. The predominant form of base stacking involves the ribosyl oxygen atom and the bases.