Publications

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.).

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

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.

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.

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.

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.

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.

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.

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 k_cat/K_m 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.

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.

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.

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.

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.

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 R_P 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 S_P enantiomer, and to determine accurately K_m and k_cat values for the individual isomers, two approaches were used to obtain the corresponding S_P and R_P 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 S_P and R_P 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 S_P configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S_P/R_P inhibition ratios of 10-100, whereas the R_P isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the S_P counterpart. Wt rePON1-induced R_P/S_P hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be 1000. The various S_P 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.

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.

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.

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.

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.

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 R_H = 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Summary: An easy-to-use, versatile and freely available graphic web server, FoldIndex

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.

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.

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.

Keywords: HALOPHILIC MALATE-DEHYDROGENASE; HALOFERAX-VOLCANII; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; STABILITY; ARCHAEON; ADAPTATION; BACTERIUM; OVEREXPRESSION; PURIFICATION

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 AChE_T tetramer.

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.

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.

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.

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 V_M = 2.65 ų Da^-1 and a solvent content of 52.7%.

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.

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.

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.

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.

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 (k_i = 2.0 M^-1 min^-1), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is > 1600-fold higher (k_i = 3300 M^-1 min^-1). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k_i = 9 × 10⁴ and 5 × 10⁵ M^-1 min^-1, respectively). Spontaneous reactivation of all four conjugates is very slow, with

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.

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.

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.

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.

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.

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.

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) ].

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.

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.

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.

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.

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.

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.

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.

(-)-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.

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.

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.

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.

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.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Pharmacology & Pharmacy

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.

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.

Keywords: Biochemistry & Molecular Biology

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.

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.

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.

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.

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.

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.

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.

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 O^γ 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.

A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of C_α 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.

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.

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 loops^2,3. 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.

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

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.

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.

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.

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 (F_o) and calculated (F_c) structure factors from X-ray diffraction patterns. The final Crystallographic discrepancy index, R = ∑|F_o-F_c|/∑F_o, 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.

Analysis of three sets of atomic coordinates of yeast phenylalanine tRNA establishes common features.

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.