Publications
2023
CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N-terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt-CCR5(8\u201320) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt-CCR5(1\u201327)(sY3,sY10,sY14) peptide cross reacts with CXCL12 and with lower KD in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt-receptor peptide binding. The Nt-CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate-binding site by an allovalency mechanism like that observed for CCL5 binding of Nt-CCR5 peptides. Nt-CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV-1 gp120 and interact with all three tyrosine/sulfated tyrosine-binding pockets of CXCL12. We suggest that the chemokine-receptors Nt-segments bind promiscuously to cognate and non-cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine-receptors' Nt-segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study.
2021
The N-terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high-affinity binding to gp120. In the present study, the interactions of gp120YU2 with a 27-residue N-terminal CCR5 peptide sulphated at position Y10 and Y14, i.e. Nt-CCR5, were studied using 13C-edited-HMQC methyl-NOESY [1H(13C)-1H], combined with transferred NOE NMR spectroscopy. A large number of pairwise interactions were observed between the methyl protons of methionine, threonine, valine and isoleucine residues of gp120, and the aromatic tyrosine-protons of Nt-CCR5. M434, V120 and V200 of gp120 were found to interact with all four tyrosine residues, Y3, sY10, sY14 and Y15. Particularly intriguing was the observation that Y3 and Y15 interact with the same gp120 methyl protons. Such interactions cannot be explained by the single cryo-EM structure of gp120/CD4/CCR5 complex published recently (Nature, 565, 318\u2013323, 2019). Rather, they are consistent with the existence of a dynamic equilibrium involving two or more binding modes of Nt-CCR5 to gp120. These different modes of binding can coexist because the surface of gp120 contains two sites that can optimally interact with a sulphated tyrosine residue and two sites that can interact favorably with a non-sulphated tyrosine residue. Modelling of gp120YU2 complexed with the Nt-CCR5 peptide or with the entire CCR5 receptor provides an explanation for the NMR observations and the existence of these different binding modes of the disordered N-terminus of CCR5. The data presented extend our understanding of the two-step model and suggest a more variable binding mode of Nt-CCR5 with gp120.
2020
The N\u2010terminal segment of the chemokine receptor CCR5, Nt\u2010CCR5, contains four tyrosine residues, Y3, Y10, Y14 and Y15. Sulfation of at least two of these tyrosine residues was found to be essential for high affinity binding of CCR5 to its chemokine ligands. Here we show that among the monosulfated Nt\u2010CCR5(8\u201020) peptide surrogates (sNt\u2010CCR5) those sulfated at Y15 and Y14 have the highest affinity for the CCL5 chemokine in comparison with monosulfation at position Y10. Sulfation at Y3 was not investigated. A peptide sulfated at both Y14 and Y15 has the highest affinity for CCL5 by up to a factor of 3, in comparison with the other disulfated (sNt\u2010CCR5) peptides. Chemical shift perturbation analysis and TRNOE measurements indicate that the sulfated tyrosine residues interact with the same CCL5 binding pocket and that each of the sulfated tyrosines at positions 10, 14 and 15 can occupy individually the binding site on CCL5 in a similar manner, although with somewhat different affinity, suggesting the possibility of allovalency in sulfated Nt\u2010CCR5 peptides. The affinity of the disulfated peptides to CCL5 could be increased by this allovalency and by stronger electrostatic interactions.
Many proteins interact with their ligand proteins by recognition of short linear motifs that are often intrinsically disordered. These interactions are usually weak and are characterized by fast exchange. NMR spectroscopy is a powerful tool to study weak interactions. The methods that have been commonly used are analysis of chemicals shift perturbations (CSP) upon ligand binding and saturation transfer difference spectroscopy. These two methods identify residues at the binding interface between the protein and its ligand. In the present study, we used a combination of transferred-NOE, specific methyl-labeling and an optimized isotope-edited/isotope-filtered NOESY experiment to study specific interactions between the 42 kDa p38α mitogen-activated protein kinase and the kinase interaction motif (KIM) on the STEP phosphatase. These measurements distinguished between residues that both exhibit CSPs upon ligand binding and interact with the KIM peptide from residues that exhibit CSPs but do not interact with the peptide. In addition, these results provide information about pairwise interactions that is important for a more reliable docking of the KIM peptide into its interacting surface on p38α. This combination of techniques should be applicable for many protein-peptide complexes up to 80 kDa for which methyl resonance assignment can be achieved.
2018
Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to similar to 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core (YU2) gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N-terminus of CCR5.
The inflammatory chemokine CCL5, which binds the chemokine receptor CCR5 in a two-step mechanism so as to activate signaling pathways in hematopoetic cells, plays an important role in immune surveillance, inflammation, and development as well as in several immune system pathologies. The recently published crystal structure of CCR5 bound to a high-affinity variant of CCL5 lacks the N-terminal segment of the receptor that is post-translationally sulfated and is known to be important for high-affinity binding. Here, we report the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated peptide corresponding to the missing first 27 residues of CCR5. Our structures show that two sulfated tyrosine residues, sY10 and sY14, as well as the unsulfated Y15 form a network of strong interactions with a groove on a surface of CCL5 that is formed from evolutionarily conserved basic and hydrophobic amino acids. We then use our NMR structures, in combination with available crystal data, to create an atomic model of full-length wild-type CCR5:CCL5. Our findings reveal the structural determinants involved in the recognition of CCL5 by the CCR5 N terminus. These findings, together with existing structural data, provide a complete structural framework with which to understand the specificity of receptor:chemokine interactions.DatabaseStructural data are available in the PDB under the accession number
Background: Tyrosine sulfation is an important post-translational modification of secreted and membrane proteins in multi-cellular organisms. This modification is catalyzed by tyrosylprotein sulfotransferases that often modify tyrosine residues in their target substrates in a heterogeneous manner. Chemokine receptors such as CCR5, which play roles in inflammation, immunity and viral infection, are sulfated on tyrosine residues in their extracellular N-termini. The heterogeneity of the sulfation has made it difficult to obtain atomic-resolution information on this region of CCR5. Homogeneously sulfated peptide surrogates can be efficiently synthesized by chemical and biochemical approaches. This communication reviews current chemical and biochemical methods for peptide tyrosine sulfation and the use of N-terminal CCR5 peptide surrogates in biochemical and structural analyses.Conclusion: Using solid phase peptide synthesis and synthons containing sulfotyrosine or sulfotyrosine neopentyl esters peptides containing up to 30 residues with multiple sulfotyrosines can be synthesized and purified in high (>50-70%) yield. Such peptides can be isotopically labeled at selected positions and used in detailed NMR investigations to investigate the interactions of sulfotyrosine residues with receptors. The application of transferred NOE studies to investigate CCL5/CCR5 interactions has led to the determination of pairwise interactions between the chemokine and its receptor.
2017
NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (KD > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the 13C-edited/13C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the 13C-edited/13C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1–27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the 13C-edited/13C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1–27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1–27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex.
V3-directed antibodies are present in practically all HIV-1 infected patients and in individuals vaccinated with gp120. The levels of maternal V3-directed antibodies were recently shown to correlate with reduced mother to child transmission, and V3 IgGs were found to be a negative correlate of risk in the RV-144 human trial. mAb directed to the tip of the V3 are capable of broad neutralization of Tier-1 and some Tier-2 viruses. Here we report an immunofocusing approach using conformationally constrained V3 peptides of different lengths. Immunofocusing with short constrained V3 peptides following immunizations with long constrained V3 peptides resulted in sera with improved neutralization of Tier-1B viruses in comparison with immunizations with the long constrained peptide alone. Immunizations only with the short constrained peptide were ineffective. Our results demonstrate that immunofocusing with constrained V3 peptides of different lengths could improve the induction of HIV-1 neutralizing antibodies.
2016
Intermolecular NOE interactions are invaluable for structure determination of biomolecular complexes by NMR and they represent the \u201cgold-standard\u201d amongst NMR measurements for characterizing interfaces. These NOEs constitute only a small fraction of the observed NOEs in a complex and are usually weaker than many of the intramolecular NOEs. A number of methods have been developed to remove the intramolecular NOEs that interfere with the identification of intermolecular NOEs. NMR experiments used to observe intermolecular NOE interactions in large protein complexes must cope with the short T2 relaxation time of the protons and heteronuclei in these complexes because they result in severe losses in sensitivity. The isotope-edited/isotope-filtered experiment is a powerful method for extraction of intermolecular NOEs in biomolecular complexes. Its application to large protein complexes is limited because of severe losses in signal-to-noise ratio caused by delays in the pulse sequence necessary for the multiple magnetization transfer steps between protons and heteronuclei. Isotope-edited/isotope-edited experiments, in which one protein is usually labeled with 13C and the other is labeled with 15N, reduce possible artifacts in the filtering experiments and improve somewhat the sensitivity of these experiments. Sensitivity can also be improved by deuteration of the components of the complex in order to replace either or both of the filtering or editing steps. Asymmetric deuteration, where aromatic residues in one protein and non-aromatic amino acids in the other are reverse protonated, can eliminate the editing and the filtering steps altogether, thus maintaining high sensitivity even for large proteins complexes. Difference spectroscopy and the use of 2D NOESY experiments without using editing or filtering steps can significantly increase the signal-to-noise ratio in experiments aimed at observing intermolecular NOEs. The measurement of NOESY spectra of three different preparations of a heterodimeric complex under investigation in which one or neither of the components is uniformly deuterated, and calculation of a double difference spectrum provides information on all intermolecular NOEs of non-exchangeable protons. Recent studies indicate that many protein-protein interactions are actually between a protein and a linear peptide recognition motif of the second protein, and determinants represented by linear peptides contribute significantly to the binding energy. NMR is a very versatile method to study peptide-protein interactions over a wide range of binding affinities and binding kinetics. Protein-peptide interactions in complexes exhibiting tight binding can be studied using single and/or multiple deuteration of the peptide residues and measuring a difference NOESY spectrum. This difference spectrum will show exclusively intra- and intermolecular interactions of the peptide protons that were deuterated. Transferred nuclear Overhauser spectroscopy (TRNOE) extends NMR to determine interactions within and between a weakly-bound rapidly-exchanging peptide and its protein target. TRNOE, together with asymmetric deuteration, is applicable to complexes up to ∼100 KDa and is highly sensitive, taking advantage of the long average T2 of the peptide protons. Among the methods described in this review, TRNOE has the best potential to determine intermolecular NOEs for the upper molecular weight limit of proteins that can be studied in detail by NMR.
Weak protein–protein and protein–ligand interactions play important roles in biological recognition. In many cases, simplification of structural studies of large protein complexes is achieved by investigation of the interaction between the protein and a weakly binding segment of its protein ligand. Detection of pairwise interactions in such complexes is a major challenge for both X-ray crystallography and nuclear magnetic resonance. We demonstrate that transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Using this approach, we revealed interactions between tyrosine residues of a 27-residue peptide (deuterated at Ile and Val residues) corresponding to the N-terminal segment of the human C-C chemokine receptor 5 (CCR5) chemokine receptor, and a 43 kDa construct of gp120 envelope protein of human immunodeficiency virus type 1 (deuterated on all aromatics) complexed with a cluster of differentiation 4-mimic miniprotein. The complex was present mostly as a dimer as determined by T2 relaxation measurements. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons, predominantly of isoleucine residues, and also of leucine and/or valine residues of gp120. The TRNOE/asymmetric deuteration method benefits from the sensitivity of the homonuclear NOESY experiment and does not suffer the sensitivity losses associated with isotope-edited/isotope-filtered approaches that rely on magnetization transfer between protons and heteronuclei that are bonded to them. The technique can be widely applied for studying large protein complexes that exhibit fast off-rates.
2015
The peptide T20, which corresponds to a sequence in the C-terminal segment of the HIV-1 transmembrane glycoprotein gp41, is a strong entry inhibitor of HIV-1. It has been assumed that T20 inhibits HIV-1 infection by binding to the trimer formed by the N-terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N- and C-terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism of HIV-1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/CD4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. The present study concludes that an amphiphilic helix on the T20 C-terminus binds through mostly hydrophobic interactions to a nonpolar gp120 surface formed primarily by the C4 region. The ten- to thousand-fold difference between the EC50 of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV-1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti-HIV therapeutics. Enfuvirtide (T20) is a peptide of the HIV-1 transmembrane glycoprotein gp41 that inhibits HIV-1 entry by interfering with the formation of the six-helix bundle by gp41. In addition, T20 was shown to bind gp120 of X4 viruses. Anglister and colleagues now report that T20 also binds to the gp120 protein of R5 viruses that cause the majority of HIV-1 infections. This binding was mapped to specific residues in T20 and the C4 region of gp120 using solution NMR techniques. The identified gp120 binding surface could be a target for anti-HIV therapeutics. This article is accompanied by a podcast, listen now. Or listen in iTunes.
C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors.
2014
The envelope spike of HIV-1, which consists of three external gp120 and three transmembrane gp41 glycoproteins, recognizes its target cells by successively binding to its primary CD4 receptor and a coreceptor molecule. Until recently, atomic-resolution structures were available primarily for monomeric HIV-1 gp120, in which the V1, V2 and V3 variable loops were omitted (gp120core), in complex with soluble CD4 (sCD4). Differences between the structure of HIV gp120core in complex with sCD4 and the structure of unliganded simian immunodeficiency virus gp120core led to the hypothesis that gp120 undergoes a major conformational change upon sCD4 binding. To investigate the conformational flexibility of gp120, we generated two forms of mutated gp120 amenable for NMR studies: one with V1, V2 and V3 omitted (mutgp120core) and the other containing the V3 region [mutgp120core(+V3)]. The TROSY-1H-15N-HSQC spectra of [2H,13C,15N]Arg-labeled and [2H,13C,15N]Ile-labeled unliganded mutgp120core showed many fewer crosspeaks than the expected number, and also many fewer crosspeaks in comparison with the labeled mutgp120core bound to the CD4-mimic peptide, CD4M33. This finding suggests that in the unliganded form, mutgp120core shows considerable flexibility and motions on the millisecond time scale. In contrast, most of the expected crosspeaks were observed for the unliganded mutgp120core(+V3), and only a few changes in chemical shift were observed upon CD4M33 binding. These results indicate that mutgp120core(+V3) does not show any significant conformational flexibility in its unliganded form and does not undergo any significant conformational change upon CD4M33 binding, underlining the importance of V3 in stabilizing the gp120core conformation. Two forms of gp120 were generated, mutgp120core and mutgp120core(+V3). The TROSY-1H-15N-HSQC spectra of unliganded mutgp120core show much fewer cross peaks than the expected number of cross peaks and in comparison with those observed for mutgp120core bound to the CD4-mimic peptide CD4M33 and both liganded and unliganded mutgp120core(+V3), suggesting that unliganded mutgp120core exhibits considerable flexibility in the millisecond time scale.
2013
Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the G-protein-coupled receptor (GPCR) superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using 1H-15N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.
Due to the different mechanisms HIV-1 has evolved to escape from a neutralizing antibody response it has been extremely challenging to develop an effective anti-HIV-1 vaccine. The V3 region of the gp120 HIV-1 envelope glycoprotein has been considered as one of the possible targets for an anti-HIV vaccine. It is well known that the V3 region of gp120 is at least partially masked in circulating strains and becomes exposed only after CD4 binding. However, when the virus is bound to surface CD4, steric hindrance prevents effective neutralization by V3-directed antibodies. Here we have used a 27-residue CD4-mimetic peptide in combination with immune sera elicited by an optimally constrained V3 peptide to enhance neutralization of a panel of clade B viruses. We observed strong synergism between the immune sera and the CD4-mimetic in the neutralization of tier 1 and a representative tier 2 clade B virus suggesting that the constrained V3 peptide immunogen correctly mimics the V3 conformation even in tier 2 clade B viruses. This synergy should improve the potential of CD4-mimetic compounds for preexposure prophylaxis and in the treatment of HIV-1-infected patients who usually manifest high titers of V3-directed antibodies. Moreover, constrained V3 immunogens elicit immune sera that may neutralize HIV in synergy with CD4 binding site antibodies that expose V3 and the coreceptor binding site.
2011
NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-alpha 2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.
Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated CCR5Tyr14 with gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.
2010
Synthetic peptides offer an attractive option for development of a V3-directed vaccine. However, immunization with flexible linear peptides may result in an immune response to multiple conformations, many of which differ from the native conformation of the corresponding region in the protein. Here we show that optimization of the location of a disulfide bond in peptides constrained to mimic the beta-hairpin conformation of the V3, yields an immunogen that elicits a 30-fold stronger HIV-1 neutralizing response in rabbits compared with the homologous linear V3 peptide. The HIV-1 neutralizing response elicited by the optimally constrained peptide is also significantly stronger than that elicited by a gp120 construct in which the V3 is exposed. Neutralization of an HIV-1 strain that shares only 72% identity with the immunizing peptide was demonstrated. The most effective immunogen was also able to neutralize primary isolates that are more resistant to neutralization such as SS1196 and 6535. (C) 2010 Elsevier Inc. All rights reserved.
Type I interferons (IFNs) make up a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNα2 using asymmetric reverse-protonation of the different complex components and two-dimensional homonuclear NOESY. This new approach revealed with an excellent signal-to-noise ratio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular mass (44 kDa). Sequential and side chain assignment of IFNAR2-EC and IFNα2 in their binary complex helped assign the intermolecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC-IFNα2 complex was calculated on the basis of the intermolecular interactions found in this study as well as four double mutant cycle constraints, previously observed NOEs between a single pair of residues and the NMR mapping of the binding sites on IFNAR2-EC and IFNα2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of intermolecular hydrogen bonds, salt bridges, and van der Waals interactions. Furthermore, our model reveals the participation of several new regions in the binding site such as the N-terminus and A helix of IFNα2 and the C domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNα2 has changed by 30° in comparison with a previously calculated model that was based on NMR mapping of the binding sites and double mutant cycle constraints. In addition, the new model strongly supports the recently proposed allosteric changes in IFNα2 upon binding of IFNAR1-EC to the binary IFNα2-IFNAR2-EC complex.
All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits, IFNAR1 and IFNAR2. In this study, we mapped the surface of IFNα2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the 1H- 15NTROSY-HSQC cross peaks of IFNα2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNα2 with IFNAR2-EC. The NMR study of the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 of 165 IFNα2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site, and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNα2 binding site for IFNAR2-EC, indicating allosteric communication between the binding sites for the two receptor subunits.
2009
Fusion of HIV-1 and target cells is mediated by the envelope protein gp41 that undergoes a series of conformational changes during the process of infection. Knowledge of the structural biology of gp41 allows the design of potent peptide inhibitors that prevent the virus from entering lymphocytes and macrophages. The design of such inhibitors is the subject of this review.
The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a β-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3IIIB peptide bound to the 0.5β antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3MN and a V3 CONSENSUS peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3CONSENSUS epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks theGPGRsegment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.
The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5β and 447-52D form β-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3 JRFL and V3 IIIB peptides bound to 447-52D and to 0.5β, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of β-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized β-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3 mn peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3 iiib peptide bound to antibody 0.5/? was assumed.
Nuclear magnetic resonance (NMR) is a very powerful tool for determining the boundaries of peptide epitopes recognized by antibodies. NMR can be used to study antibodies in complexes that exhibit a wide range of binding affinities from very weak and transient to very tight. Choice of the specific method depends upon the dissociation constant, especially the ligand off-rate. Epitope mapping by NMR is based on the difference in mobility between the amino acid residues of a peptide antigen that interact tightly with the antibody and residues outside the epitope that do not interact with the antibody. The interacting peptide residues become considerably immobilized upon binding. Their mobility will resemble that of the antibody's residues. Several NMR methods were developed based on these characteristics. In this chapter we discuss some of these methods, including dynamic filtering, comparison of H-1-N-15 HSQC peaks' intensities, transverse relaxation time, measurements of H-1-N-15 nuclear Overhauser effect (NOE) values, and measurements of T-1p relaxation time.
2008
The HIV-1 envelope glycoprotein gp41 undergoes a sequence of extensive conformational changes while participating in the fusion of the virus with the host cell. Since the discovery of its postfusion conformation, the structure and function of the protease-resistant six-helix bundle (6-HB) have been the subject of extensive investigation. In this work, we describe additional determinants (S528-Q540 and W666-N677) in the fusion peptide proximal region (FP-PR) and the membrane proximal external region (MPER) that stabilize the six-helix bundle and are involved in the interaction of T-20 (FUZEON, an anti-HIV-1 fusion inhibitor drug) with the gp41 FP-PR. Circular dichroism and sedimentation equilibrium measurements indicate that the 1:1 mixture of N\u2032 and C\u2032 peptides comprising residues A541-T569 and I635-K665 from the gp41 first and second helical repeats, HR1 and HR2, respectively, fail to form a stable six-helix bundle. Triglutamic acid and triarginine tags were added to these N\u2032 and C peptides, respectively, at the termini distant from the FP-PR and the MPER to alter their pi and increase their solubility at pH 3.5. The tagged HR1 and HR2 peptides were elongated by addition of residues S528-Q540 from the FP-PR and residues W666-N677 from the MPER, respectively. A 1:1 complex of the elongated peptides formed a stable six-helix bundle which melted at 60°C. These results underscore the importance of a detailed high-resolution characterization of MPER interactions, the results of which may improve our understanding of the structure-function relationship of gp41 and its role in HIV-1 fusion.
Analysis of V3 and C4 sequences of HIV-1 reveals correlated mutations at gp120 positions 322 and 440, and a very strong preference for a positively charged residue at position 440 when position 322 is negatively charged. This observation suggests that these two residues are close to each other and interact electrostatically in R5 viruses. This interaction was used to model V3 in the context of gp120 using NMR data for the V3 loop and the crystal structure of the gp120-core. The interaction between residues 322 and 440 may serve as part of the molecular switch for HIV-1 phenotype conversion.
Voltage-gated sodium channels (Na(v)s) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Na(v)s with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion a.-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In-this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Na-v was mapped on the highly insecticidal cc-neurotoxin, Lqh alpha IT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide H-1 and N-15 chemical shifts upon binding. This analysis suggests that the five-residue turn (residues (Lq)K8-(Lq)C12) of Lqh alpha IT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues (Lq)R18, (Lq)W38, and (Lq)A39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, (Lq)V13, was identified as being important for Na-v binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of (Lq)V13 to Nav binding was further verified by mutagenesis. Future studies involving other extracellular regions of Na(v)s are required for further characterization of the structure of the Lqh alpha IT-Na(v)s binding site.
2007
Keywords: ALTERNATIVE CONFORMATIONS
2006
HIV-1 coreceptor usage plays a critical role in virus tropism and pathogenesis. A switch from CCR5- to CXCR4-using viruses occurs during the course of HIV-1 infection and correlates with subsequent disease progression. A single mutation at position 322 within the V3 loop of the HIV-1 envelope glycoprotein gp120, from a negatively to a positively charged residue, was found to be sufficient to switch an R5 virus to an X4 virus. In this study, the NMR structure of the V3 region of an RS strain, HIV-1JR-FL, in complex with an HIV-1-neutralizing antibody was determined. Positively charged and negatively charged residues at positions 304 and 322, respectively, oppose each other in the β-hairpin structure, enabling a favorable electrostatic interaction that stabilizes the postulated R5 conformation. Comparison of the R5 conformation with the postulated X4 conformation of the V3 region (positively charged residue at position 322) reveals that electrostatic repulsion between residues 304 and 322 in X4 strains triggers the observed one register shift in the N-terminal strand of the V3 region. We posit that electrostatic interactions at the base of the V3 β-hairpin can modulate the conformation and thereby influence the phenotype switch. In addition, we suggest that interstrand cat ion-π interactions between positively charged and aromatic residues induce the switch to the X4 conformation as a result of the S306R mutation. The existence of three pairs of identical (or very similar) amino acids in the V3 C-terminal strand facilitates the switch between the R5 and X4 conformations.
The V3 loop is part of the gp120 glycoprotein, an extracellular protein located on the membrane of the human immunodeficiency virus (HIV-1). This loop is significantly important in many biological processes of the virus and contains the principal neutralizing determinant (PND). The PND is one of the most variable regions of the envelope, and this is probably related to the ability of the HIV virus to escape the immunologic defenses of the target host. Particular attention has been paid to the central part of the V3 loop which contains a highly conserved GPGR/GPGQ sequence and represents the binding site for antibodies. Many attempts have been made to design synthetic peptides as mimics of the V3 loop capable of eliciting immune response. However, this strategy suffers from the great conformational flexibility small peptides have in solution, which together with bioavailability represents the most important limitation to the usefulness of synthetic peptides as drugs and as synthetic immunogens. The use of conformationally constrained peptides can alleviate this problem. Early works using NMR studies have shown that a V3IIIB loop-derived peptide is conformationally heterogeneous when free in water. Upon complexation with 0.5β, a monoclonal neutralizing antibody specific for the HIV-1IIIB strain, it adopts a β-hairpin conformation with the central proline forming a type VIb β-turn. In this study, we report the design and characterization of a conformationally restricted peptide with a sequence identical to that previously described, but with thiazolidine derivatives replacing the proline. The affinity of the 2,2-dimethylthiazolidine derivative for 0.5β demonstrates that this moiety can successfully be used to mimic the proline in a cis conformation. This peptide not only displays a high propensity to adopt a β-hairpin conformation but also retains the type VIb RGPG β-turn similar to that found in the native complex. These compounds could help in elaborating more efficient immunogens for HIV-1 synthetic vaccine development.
Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFNα2 with R2-EC using multidimensional NMR techniques. NMR shows that IFNα2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFNα2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Å, and its well-defined orientation between IFNα2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand-receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules. Published by Cold Spring Harbor Laboratory Press.
2005
The HIV-1 envelope glycoprotein gp41 is responsible for viral fusion with the host cell. The fusion process, as well as the full structure of gp41, is not completely understood. One of the strongest inhibitors of HIV-1 fusion is a 36-residue peptide named T-20, gp41(638-673) (Fuzeon, also called Enfuvirtide or DP-178; residues are numbered according to the HXB2 gp160 variant) now used as an and HIV-1 drug. This peptide also contains the immunogenic sequences that represent the full or partial recognition epitope for the broadly neutralizing human monoclonal antibodies 2F5 and 4E10, respectively. Due to its hydrophobicity, T-20 tends to aggregate at high concentrations in water, and therefore the structure of this molecule in aqueous solution has not been previously determined. We expressed a uniformly 13C/ 15N-labeled 42-residue peptide NN-T-20-NITN (gp41636-677) and used heteronuclear 2D and 3D NMR methods to determine its structure. Due to the additional gp41-native hydrophilic residues, NN-T-20-NITN dissolved in water, enabling for the first time determination of its secondary structure at near physiological conditions. Our results show that the NN-T-20-NITN peptide is composed of a mostly unstructured N-terminal region and a helical region beginning at the center of T-20 and extending toward the C-terminus. The helical region is found under various conditions and has been observed also in a 13-residue peptide gp41659-671. We suggest that this helical conformation is maintained in most of the different tertiary structures of the gp41 envelope protein that form during the process of viral fusion. Accordingly, an important element of the immunogenicity of gp41 and the inhibitory properties of Fuzeon may be the propensity of specific sequences in these polypeptides to assume helical structures.
The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is involved in gp120 binding to the chemokine receptors CCR5 and CXCR4, which serve as co-receptors in HIV-1 infection. The sequence of V3 determines whether the virus binds to CCR5 and infects predominantly macrophages (R5 virus) or to CXCR4 and infects mostly T-cells (X4 virus). This review summarizes structural information for V3 peptides in complex with HIV-1 neutralizing antibodies. Nuclear magnetic resonance studies of the V3 peptides led to the proposal of a mechanism for co-receptor selectivity. Experiments to further explore this mechanism and potential applications of V3 structural information are discussed.
A method for the measurement of proton T1ρ relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90deg; excitation in a HOHAHA pulse sequence. The experiment is applied to α-bungarotoxin (α-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) α-subunit. A good correlation between high T1ρ values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the α-BTX complex with an AChR peptide and the model for α-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor α-subunit is exhibited, while residues implicated in binding γ- and δ-subunits retain their mobility. In addition, the quantitative T1ρ measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.
Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5β, raised against gp120 of the X4 isolate HIV-1IIIB, neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3MN and V3IIIB, adopt alternative β-hairpin conformations when bound to 447-52D and 0.5β, respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3IIIB peptide bound to 447-52D was determined by NMR. V3IIIB and V3MN peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3IIIB peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5β. Comparison of the three known V3 structures leads to a model in which a 180° change in the orientation of the side chains and the resulting one-residue shift in hydrogen bonding patterns in the N-terminal strand of the β-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, 1314, and 1316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5β with variable residues underlie its strain specificity.
2004
The antiviral and anti proliferative activities of type I interferons (IFNs) are mediated by a common receptor, and its second subunit (IFNAR2) exhibits nanomolar affinity to both IFNalpha and IFNbeta subtypes. We have previously determined the structure of the IFN-binding extracellular domain of IFNAR2 (IFNAR2-EC) using multidimensional NMR [Chill, J. H., Quadt, S. R., Levy, R., Schreiber, G. E., and Anglister, J. (2003) Structure 11, 791-802], showing it to comprise two fibronectin domains linked by a hinge. As the first cytokine receptor structure determined in the unliganded state and in solution, IFNAR2EC offers an opportunity to characterize the dynamics of the cytokine receptor family and their correlation to biolooical function. Backbone dynamics of IFNAR2-EC were investigated using N-15 relaxation at 11.74 and 18.79 T, and measurements of residual dipolar couplings (RDCs). Dynamics of the binding site distinguish between rigid structural domains, which stabilize the binding site conformation, and a more flexible binding interface which interacts with the ligand. Measurements of diffusional anisotropy and RDCs and model-free analysis all show that the backbone of the hinge interdomain region of IFNAR2EC is rigid on the picosecond to nanosecond time scale. Signal transduction in cytokines receptors is initiated by ligand-induced juxtaposition of the two receptor subunits, triggering the mutual phosphorylation of kinases associated to their cytoplasmic domains. The rigidity of the hinge ensures correct positioning of the receptor subunits in the ternary signaling complex and modulates the interaction between kinases in the cytoplasm, thereby controlling the rate and efficiency of phosphorylation.
2003
The potent antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by a single receptor comprising two subunits, IFNAR1 and IFNAR2. The structure of the IFNAR2 IFN binding ectodomain (IFNAR2-EC), the first helical cytokine receptor structure determined in solution, reveals the molecular basis for IFN binding. The atypical perpendicular orientation of its two fibronectin domains explains the lack of C domain involvement in ligand binding. A model of the IFNAR2-EC/IFNα2 complex based on double mutant cycle-derived constraints uncovers an extensive and predominantly aliphatic hydrophobic patch on the receptor that interacts with a matching hydrophobic surface of IFNα2. An adjacent motif of alternating charged side chains guides the two proteins into a tight complex. The binding interface may account for crossreactivity and ligand specificity of the receptor. This molecular description of IFN binding should be invaluable for study and design of IFN-based biomedical agents.
The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, VL and VH, respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed VH and VL were constructed. A pe1B signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pe1B sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the VL and VH were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the VH and the VL chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The VL of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.
The V3 loop of the HIV-1 envelope glycoprotein gp120 is involved in binding to the CCR5 and CXCR4 coreceptors. The structure of an HIV-1MN V3 peptide bound to the Fv of the broadly neutralizing human monoclonal antibody 447-52D was solved by NMR and found to be a β hairpin. This structure of V3MN was found to have conformation and sequence similarities to β hairpins in CD8 and CCR5 ligands MIP-1α, MIP-1β, and RANTES and differed from the β hairpin of a V3IIIB peptide bound to the strain-specific murine anti-gp120IIIB antibody 0.5β. In contrast to the structure of the bound V3MN peptide, the V3IIIB peptide resembles a β hairpin in SDF-1, a CXCR4 ligand. These data suggest that the 447-52D-bound V3MN and the 0.5β-bound V3IIIB structures represent alternative V3 conformations responsible for selective interactions with CCR5 and CXCR4, respectively.
2002
The peptide gp41659-671 (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41659-671 in water. This peptide forms a monomeric 310-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 310-helix is confirmed by its characteristic CD pattern. Studies of the 310-helix have been hampered by the absence of a model peptide adopting this conformation, gp41659-671 can serve as such a model to investigate the spectral characteristics of the 310-helix, the factors that influence its stability, and the propensity of different amino acids to form a 310-helix. The observation that the 310-helical conformation is highly populated in the peptide gp41659-671 indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41659-671 segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41659-671 is an autonomous folding unit, peptide immunogens consisting of the complete gp41659-671 sequence are likely to induce antibodies highly cross-reactive with HIV-1.
The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor α1 subunit in complex with α-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in α-bungarotoxin binding. One face of the long β hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the β hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two α-bungarotoxin molecules shows that this pocket is occupied by the conserved α-neurotoxin residue R36, which forms cation-π interactions with both αW149 and γW55/δW57 of the receptor and mimics acetylcholine.
The a subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for α-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle α subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that α211, the entire amino-terminal extracellular domain of AChR α subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The α211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mM (∼30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with α-bungarotoxin (kD 0.2 nM) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of α211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently 15N-labeled in P. pastoris, and proton crosspeaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.
The human interferon receptor (IFNAR) mediates the antiviral and antiproliferative activities of type I interferons (IFNs). This receptor is comprised of subunits IFNAR1 and IFNAR2, the latter exhibiting nanomolar affinity for IFNs. Here the extracellular domain of IFNAR2 (IFNAR2-EC), a soluble 25 kDa IFN-binding polypeptide, and its complex with IFN-α2 were studied using multidimensional NMR. IFNAR2-EC is comprised of two fibronectin-III (FN-III) domains connected by a helical hinge region. The deduced global fold was utilized to improve the alignment of IFNAR2-EC against structurally related receptors and to model its structure. A striking feature of IFNAR2-EC is the limited and localized deviations in chemical shifts exhibited upon ligand binding, observed for only 15% of its backbone 1H and 15N nuclei. Analysis of these deviations maps the IFN-α2 binding site upon IFNAR2-EC to a contiguous surface on the N-terminal domain, including the S3-S4 loop (residues 44-53), the S5-S6 loop and S6 β-strand (residues 74-82), and the S7 β-strand and the hinge region (residues 95-105). The C-terminal domain contributes only marginally to ligand binding, and no change in the hypothesized interdomain interface is observed. The proposed binding domain encompasses all residues implicated by mutagenesis studies in IFN binding, and suggests adjacent residues cooperate in forming the binding surface. D2O-exchange experiments indicate that binding of IFN-α2 induces tightening of the N-terminal domain of IFNAR2-EC. This increase in receptor rigidity may play an important role in initiating the intracellular stage of the IFN signaling cascade.
Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression system is generally applicable for biosynthesis of V3-related peptides and was also used to prepare the V3JR-FL. The 447-52D Fab fragment was obtained by a short enzymatic papain cleavage of the whole antibody. Preliminary NMR spectra demonstrate that full structural analysis of the V3MN complexed to the 447-52D Fab is feasible. This system enables studies of the same epitope bound to different HIV-1 neutralizing antibodies.
2001
The α-subunit of the nicotinic acetylcholine receptor (αAChR) contains a binding site for α-bungarotoxin (α-BTX), a snake-venom-derived α-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of αAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major αAChR determinant interacting with α-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the αAChR were complexed with α-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to α-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T2 measurements. Complete resonance assignment of the 11.2 kDa complex of α-BTX bound to the αAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both α-BTX and the bound αAChR peptide were determined using 2D 1H NMR experiments. The peptide folds into a β-hairpin conformation, in which residues RH186-RV188 and RY198-RD200 form the two β-strands. Residues RY189-RT191 form an intermolecular β-sheet with residues BK38-BV40 of the second finger of α-BTX. These results accurately pinpoint the α-BTX-binding site on the αAChR and pave the way to structure determination of this important αAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.
2000
Continued use of non-specific chemical insecticides poses potential risks to the environment and to human health resulting from non-target toxicity and increased insect resistance to these agents. Scorpions produce anti-insect selective polypeptide toxins that bind to and modulate voltage- sensitive ion channels in excitable tissues, thus offering alternative, environmentally safer means for insect pest control. Despite this potential, little is known about their structural elements dictating anti-insect preference, which may be useful for the design of selective insecticides. We used a bacterial system for expression and genetic dissection of two pharmacologically distinct scorpion toxins: alpha and excitatory. By exploiting a multi-disciplinary approach consisting of mutagenesis, protein chemistry, electrophysiology, binding and toxicity assays, and structural studies, we elucidated the bioactive surface of two anti-insect toxins, LqhαIT and Bj-xtrIT. In both polypeptides the bioactive surface is composed of residues surrounding the C-terminal region. In addition, a direct, immediate approach in using the toxin genes was demonstrated by engineering baculoviruses with cDNAs encoding LqhIT2 (depressant toxin), and LqhIT1 (excitatory toxin) resulting in viral vectors with significantly improved insecticidal efficacy. (C) 2000 Society of Chemical Industry.
Background: The protein 0.5β is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1 (IIIB) strain. The epitope recognized by 0.5β is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. Results: Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5β in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5β Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5β encompasses 14 of the 18 P1053 residues. The bound peptide assumes a β-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 Å and 743 Å respectively, in the 0.5β Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. Conclusions: The surface of P1053 that is in contact with the anti-protein antibody 0.5β is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5β antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the β-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.
We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp 120 antibody 0.5β, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the η nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5β complex. These results confirm the antibody- dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of φ and ψ backbone dihedral angles in the RP135/0.5β complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.
The refined solution structure of a peptide representing the full epitope of the HIV-1IIIB V3 loop in complex with the anti-gp120 antibody Fv fragment was determined using isotope-filtered and isotope-edited NMR. Both the 15N-labeled peptide in complex with the unlabeled Fv and the unlabeled peptide complexed with the uniformly 15N, 13C-labeled Fv were investigated. The backbone of the bound peptide adopts a well defined b-hairpin conformation with two twisted anti-parallel β-strands linked by a type VI tight turn comprising residues RGPG. The central glycine and proline residues of the turn are linked by a cis peptide bond. 15N{1H} NOE measurements demonstrated that the backbone of the bound peptide including the central QRGPGR loop is well ordered in the bound state. The V3 loop peptide solution structure is significantly different from the peptide conformation in the X-ray structures of three anti-peptide antibody/V3MN peptide complexes. These differences seem to be dictated by the antibody dependence and HIV strain-specificity of the V3 peptide fold.
The 0.5β monoclonal antibody is a very potent strain-specific HIV- neutralizing antibody raised against gp120, the envelope glycoprotein of HIV- 1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody-peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5β, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the β-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. and Anglister, J. (1999) Nat. Struct. Biol. 6, 331- 335].
1999
'Melittin, a 26 residue, non-cell-selective cycolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V-5,V-8,I-17,K-21-melittin, D-amino acids at positions V-5,V-8,I-17,K-21) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V-5 8,I-17,K-21-melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]-V-5,V-8,I-17,K-21-melittin binds strongly and destabilizes only negatively charged phospholipid vesicles, in contrast to native melittin, which binds strongly also zwitterionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D]-V-5,V-8,I-17,K-21-melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were-done with both melittin and [D]-V-5,V-8,I-17,K-21-melittin. The structure of the diastereomer was characterized by NMR in water, as well as in three different membrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, methanol, and dodecylphosphocholine/phosphatidylglycerol (DPC/DMPG) micelles. The NMR data revealed an amphipathic alpha-helix only in the C-terminal region of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V-5,V-8,I-17,K-21-melittin are oriented parallel to the membrane surface. This study indicates the role of secondary structure formation in selective cytolytic activity of [D]-V-5,V-8 ,I-17,K-21- melittin. While the N-terminal helical structure is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural element for binding and insertion into zwitterionic membranes and for hemolytic activity.
The refined solution structure of an 18-residue HrV-1(IIIB) V3 peptide in complex with the Fv fragment of an anti-gp120 antibody reveals an unexpected type VI beta-turn comprising residues RGPG at the center of a beta-hairpin. The central glycine and proline of this turn are linked by a cis peptide bond. The residues of the turn interact extensively with the antibody Fv. N-15{H-1} NOE measurements show that the backbone of the peptide, including the central QRGPGR loop, is well ordered in the complex. The solution structure is significantly different from the X-ray structures of HIV-1(MN) V3 peptides bound to anti-peptide antibodies. These differences could be due to a two-residue (QR) insertion preceding the GPGR sequence in the HIV-1(IIIB) strain, and the much longer peptide epitope immobilized by the anti-gp120 antibody.
Solid-state NMR measurements have been carried out on frozen solutions of the complex of a 24-residue peptide derived from the third variable (V3) loop of the HIV-1 envelope glycoprotein gp120 bound to the Fab fragment of an anti-gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody-bound state. In combination with earlier crystal structures of V3 peptide-antibody complexes and existing data on the cross-reactivity of the antibodies, the solid-state NMR measurements suggest that the Gly-Pro-Gly- Arg (GPGR) motif adopts an antibody-dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full-length gp 120. These measurements are the first application of solid-state NMR methods in a structural study of a peptide-protein complex.
NMR spectroscopy has become an important tool for structural studies of proteins and macromolecular complexes. In case of an antibody-antigen complex, different approaches are taken to alleviate the size limit, which is beyond NMR capabilities. The method of choice depends on the kinetic properties of the system under investigation and on the antibody fragment available. We have recently studied the conformation of RP135, a 24-residue HIV-1 peptide corresponding to the principal neutralizing determinant of the virus envelop glycoprotein gp120, in complex with 0.5 beta, a neutralizing antibody raised against gp120. The binding of the peptide to the antibody was too strong to observe TRNOE, therefore 2D-NOESY difference spectroscopy was applied using three strategies: (a) deuteration of specific residues of the peptide; (b) Arg-->Lys replacement and; (c) truncation of the peptide antigen. The restraints on interproton distances within the bound peptide were used to calculate its conformation. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form antiparallel beta-strands.
1998
RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser6 - Thr19 are part of the epitope, while Lys5 and Ile20 are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.
1997
The principal neutralizing determinant (PND) of human immunodeficiency virus type 1 (HIV-1) is located in the third hypervariable region (V3) of the virus envelope glycoprotein gp120. The conformation of a V3 peptide of HIV- l(IIIB) bound to the Fab fragment of an anti-gp 120 HIV neutralizing antibody, 0.5/β, was studied by 1H NMR spectroscopy. This 18-residue peptide represents the epitope recognized by 0.5β and encompasses most of the PND. The slow off-rate of the peptide prevents the observation of peptide/Fab interactions as well as intramolecular interactions within the bound peptide by transferred nuclear Overhauser enhancement (TRNOE). To detect and assign interactions within the bound peptide in the 52 kDa complex, NOESY difference spectra were measured using three strategies: (a) deuteration of peptide residues, (b) Arg → Lys replacements, and (c) truncation of the peptide antigen. Each difference spectrum was calculated between NOESY spectra measured for two Fab complexes in which the bound peptides differed in their deuteration or in their sequence. The difference spectra revealed numerous interactions between the N-terminus of the epitope (Arg-4, Lys-5, Ser-6, Ile-7, and Ile-9) and its C-terminus (Phe-17, Val-18, Thr-19, and Ile-20). The assigned NOE interactions within the bound peptide were translated into distance restraints that were used to calculate the conformation of the bound peptide by the hybrid distance geometry/simulated annealing method. A total of 39 long-range (residues i - j > 4), 14 short- range, and 69 intraresidue NOE interactions within the bound peptide have been assigned. Twelve structures without NOE constraint violations were obtained, having a 1.6 Å rms deviation for the backbone atoms. The peptide forms a 10-residue loop, while the two segments flanking this loop, KSI and VTI, interact extensively with each other and possibly form antiparallel β- strands. This loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.
The solution structure of the complex between α-bungarotoxin (α-BTX) and a 13-residue library-derived peptide (MRYYESSLKSYPD) has been solved using two-dimensional proton-NMR spectroscopy. The bound peptide adopts an almost-globular conformation resulting from three turns that surround a hydrophobic core formed by Tyr-11 of the peptide. The peptide fills an α- BTX pocket made of residues located at fingers I and II, as well as at the C- terminal region. Of the peptide residues, the largest contact area is formed by Tyr-3 and Tyr-4. These findings are in accord with the previous data in which it had been shown that substitution of these aromatic residues by aliphatic amino acids leads to loss of binding of the modified peptide with α-BTX. Glu-5 and Leu-8, which also remarkably contribute to the contact area with the toxin, are present in all the library-derived peptides that bind strongly to α-BTX. The structure of the complex may explain the fact that the library-derived peptide binds α-BTX with a 15-fold higher affinity than that shown by the acetylcholine receptor peptide (α185-196). Although both peptides bind to similar sites on α-BTX, the latter adopts an extended conformation when bound to the toxin [Basus, V., Song, G. and Hawrot, E. (1993) Biochemistry 32, 12290-12298], whereas the library peptide is nearly globular and occupies a larger surface area of α-BTX binding site.
The solution structure of a recombinant active α-neurotoxin from Leiurus quinquestriatus hebraeus, LqhαIT, was determined by proton two- dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion α-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 Å. These structures had root mean square deviations of 0.49 and 1.00 Å with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of LqhαIT consists of an α-helix, a three-strand antiparallel β- sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a 'herringbone' arrangement. Positive φ angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1- 5 s-1 at 308 K. The conformation of the polypeptide backbone of LqhαIT is very similar to that of the most active antimammalian scorpion α-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified LqhαIT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of LqhαIT.
1996
The construction, expression, and purification of an active Fv fragment of the 0.5β monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1(IIIB) was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311,315, and 318 in gp120 of HIV-1(IIIB)) reduce the binding free energy by 0.22 (± 0.20), 4.32 (± 0.16), and 1.58 (± 0.17) kcal mol-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (± 0.22), -0.55 (± 0.18), and -0.97 (± 0.19) kcal mol-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.
1995
Triple resonance 3D NMR methods have been used to study the interaction between calcineurin B and a peptide fragment of calcineurin A for which it has high affinity (KD ∼4 × 10-7 M). Although calcineurin B aggregates at NMR concentrations of ∼ 1 mM, in the presence of a target peptide fragment of calcineurin A it becomes monomeric and yields NMR spectra that are very similar to those reported previously for calcineurin B solubilized by the zwitterionic detergent CHAPS. Changes in chemical shifts between CHAPS- and peptide-solubilized calcineurin B are small which is indicative of no differences in secondary structure. Residues most affected by binding to target peptide are found primarily on the hydrophobic faces of the four helices, present in each of the two globular domains in calcineurin B, and in the loops connecting helices II and III, IV and V, and possibly in the C-terminal 12 residues, which also exhibit a change in mobility.
The interactions of the peptide RP135a (RKSIRIQRGPGRAFVT), corresponding to residues 311-326 of gp120 of HIV-1IIIB, with the anti-gp120 HIV-1IIIB neutralizing antibody 0.5β were studied by NMR. The NOESY difference spectra measured using specifically deuterated derivatives of the peptide show exclusively the interactions of the deuterated residues both within the bound peptide and with the Fab fragment of the antibody. These measurements reveal hydrophobic interactions within the bound peptide between Ile-4, Ile-6 and Val-15 that create a 12-residue loop with these residues at the base and the conserved GPGR sequence at its top.
Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat\u2010labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50\u201364 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor\u2010bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a β\u2010strand to the central α\u2010helix. Ser 55 and Gin 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gin 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino\u2010terminal part of CTP3 forms a β\u2010turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR\u2010derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross\u2010reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT.
The 24‐amino‐acid peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) corresponds in its amino acid sequence to the principal neutralizing determinant of the human immunodeficiency virus type‐1, IIIB isolate (HIV‐1IIIB, residues 308– 331 of the envelope glycoprotein gp120). In order to map the antigenic determinant recognized by 0.5β, the complex of RP135 with an anti‐gp120 HIV neutralizing antibody, 0.5β, which cross reacts with the peptide, was studied by using two‐dimensional NMR spectros‐copy. A combination of homonuclear Hartmann Hahn two‐dimensional experiment and rotating‐frame Overhauser enhancement spectroscopy of the Fab/peptide complex measured in H2O was used to eliminate the resonances of the Fab and the tightly bound peptide residues and to obtain sequential assignments for those parts of the peptide which retain considerable mobility upon binding. In this manner, a total of 14 residues (Ser6–Thrl9) were shown to be part of the antigenic determinant recognized by the antibody 0.5β. Lys5 and Ile20 were found to retain considerable mobility in the bound peptide while their amide protons undergo significant change in chemical shift upon binding. This observation suggests that these two residues are at the boundaries of the determinant recognized by the antibody. Competitive binding experiments using truncated peptides strongly support the NMR observations.
A spin-labeled peptide antigen (TEMPO-VEVPGSQHIDSQ) was used to measure NOESY difference spectra that show interactions in the binding site region of the Fab fragment of the anti-cholera toxin pep-tide antibody TE33. In addition to identification of peptide-Fab interactions and interactions within the bound peptide, these difference spectra show well-resolved cross peaks due to interactions within the large Fab fragment (50 kDa). These difference spectra indicate that the conformational changes in the Fab upon peptide binding are confined to the combining site region of the antibody. The NOESY difference spectra of selectively deuterated Fab molecules were used in combination with HOHAHA measurements to assign the interactions to amino acid type and to identify the interactions within the Fab as either inter- or intraresidue interactions. The assignment of interactions within the Fab to corresponding aromatic residues in the Fab sequence was facilitated by an earlier NMR-derived model calculated on the basis of NOE restraints on Fab-peptide and intra-bound-peptide distances. The new restraints on distances within the Fab, combined with the previously obtained restraints, were used to generate a refined NMR-derived model for the TE33- peptide complex.
To increase our understanding of the molecular basis for antibody specificity and for the cross‐reactivity of antipeptide antibodies with native proteins, it is important to study the three‐dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody–peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab–peptide complex (Fab: antibody fragment made of Fv—the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen—the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two‐dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3). which differ in their cross‐reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. © 1994 John Wiley & Sons, Inc.
1994
The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13Cα and 13Cβ secondary chemical shifts, J(HNHα) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119–174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.
1993
To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of anti-peptide antibodies with native proteins it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen and the bound peptide conformation. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the information concerning antibody-peptide interactions and intramolecular interactions in the bound ligand from the crowded and unresolved spectrum of the Fab complex. These measurements yield restraints on interproton distances in the complex which are used to dock the peptide into calculated models for the antibodies' combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3), which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by antibodies, the structure of their combining sites and relationships between antibodies' primary structure, and their interactions with pep tide antigens.
Transferred nuclear Overhauser effect (TRNOE) spectroscopy can be used to study intra- and intermolecular interactions of bound ligands complexed with large proteins. However, the 2D NOE (NOESY) spectra of large proteins are very poorly resolved and it is very difficult to discriminate the TRNOE cross peaks, especially those due to intermolecular interactions, from the numerous cross peaks due to intramolecular interactions in the protein. In previous studies we measured two-dimensional difference spectra that show exclusively TRNOE and exchange cross-peaks (Anglister, J., 1990. Quart. Rev. Biophys. 23:175\u2013203). Here we show that a filtering method based on the difference between the T1rho values of the ligand and the protein protons can be used to directly obtain a two-dimensional transferred NOE spectrum in which the background cross-peaks due to intramolecular interactions in the protein are very effectively removed. The usefulness of this technique to study protein ligand interactions is demonstrated for two different antibodies complexed with a peptide of cholera toxin (CTP3). It is shown that the T1 rho-filtering alleviates t problems encountered in our previous measurements of TRNOE by the difference method. These problems were due to imperfections in the subtraction of two spectra measured for two different samples.
1992
Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a β-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a β-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a β-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than a-helix or β-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function, (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen, (b) Residues 31, 31c, and 3le of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen, (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.
The 24 amino acid peptide RP135 corresponds in its amino acid sequence to the principal neutralizing determinant (PND) of the IIIB isolate of HIV-1. Although the sequence of the PND is highly variable, its central part, containing the sequence GPGR, is conserved in most HIV isolates. Using 2D NMR and CD spectroscopy, we have studied the conformation of RP135 and of two shorter versions : one (P547) that includes the GPGR sequence with the N-terminal part of the peptide and the other (P344) that includes GPGR and the C-terminal segment of RP135. In water, the C-terminal part of RP135 was found to exist in several transient turnlike conformations (“nascent helix”). A helical conformation was found to be stabilized by the addition of TFE. A transient turn was observed also in the GPGR sequence, both in water and in aqueous TFE solutions. While no nascent helix conformations could be observed in the N-terminal part of RP135 in water, a helical conformation was partially stabilized by the addition of TFE. The conformations of the two shorter versions of the peptide were similar to those of the corresponding parts of RP135, except that the transient turn in GPGR could not be detected in P547 dissolved in water. The turn in GPGR was previously predicted by Larosa et al. (1990) and was observed by Chandrasekhar et al. (1991) in the PND peptide of HIV-1MN (RP142), which shares only 56% identity with RP135. However, nascent helix conformations were not observed in aqueous solutions of RP142. It is possible that the mutations, deletions, and insertions in the PND peptides modulate their tendency to form nascent helix conformations in water, without affecting the formation of a turn in the conserved GPGR sequence.
1991
This chapter describes an approach to study ligand–protein interaction using two-dimensional (2D) transferred nuclear Overhauser effect (NOE) difference spectroscopy. The methodology developed in laboratory allows obtaining detailed information concerning the interactions among residues in a peptide antigen and residues in the combining site of a monoclonal antibody. Transferred NOE difference spectroscopy, in conjunction with the specific deuteration of the antibody, provides parameters that give information about distances among the above residues. Unfortunately, the number of NOE cross-peaks is not sufficient to calculate an unequivocal structure for the bound antigen. The comparison of nuclear magnetic resonance (NMR) data with calculated models allows to examine the validity of a given model and to exclude bad models that result from choosing improper segments from the known three-dimensional structures of other antibodies. In all cases to date, information forthcoming from such studies requires comparison with crystal structures to rationalize interactions that are apparent in the NMR spectra.
1990
The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The VH sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a β-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 Å2, about half of the contact area observed in protein-antibody complexes.
The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy. The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA). Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin. The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide. Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA). These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody. The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide. Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide. In the bound peptide, we observe interactions of a lysine residue with aspartate-10 β protons. While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide. The strong interaction of TE34 with the negatively charged C-terminus of CTP3 is one of the main reasons for its lack of cross-reactivity with the native toxin. Similar use of modified peptides may extend the applicability of 2D TRNOE difference spectrscopy to the study of other antibody-peptide complexes involving slow peptide off-rates.
1989
The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE), Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50000.
The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specincities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.
1988
Specifically deuteriated Fab fragments of the anti-spin-label antibody AN02 were prepared. NMR difference spectra were obtained, in which the spectrum of Fab with some fraction of the binding sites occupied with spin-label hapten was subtracted from the spectrum of Fab with no spin-label. the peak heights were analyzed as a function of the fractional occupation of the binding site, using a computer program that calculates a best fit to the observed spectra. This method treats all of the peaks in the spectra simultaneously. Analyzing all peaks at once allows for the interdependencies in the spectra arising from overlap of positive and negative signals from different peaks. the fitting program calculates line widths for the peaks arising from protons in the binding site region. Almost all of the line widths calculated for the spectrum of the Fab complex with diamagnetic hapten dinitrophenyldiglycine were found to be narrower than the line widths of the corresponding resonances in the spectrum of Fab with an empty binding site. the distances of the binding site region protons from the unpaired electron of the hapten were also obtained from this calculation. Two tyrosine protons were found to be close (
The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR). The synthetic peptide is CTP3 (residues 50–64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera. The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin. The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin. Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids. The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34. By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues. The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine. In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide. No magnetization transfer was observed between the tryptophan residues of TE34 and the histidine residue of the peptide. The involvement of a tryptophan residue in the combining site of TE33 and TE32 is further demonstrated by the considerable quenching of antibody fluorescence observed upon the binding of the peptide. Such quenching is not observed in TE34. The observed differences between these two types of monoclonal antibodies may be relevant to the complementarity of their respective binding sites to the conformation of the CTP3 peptide in the native toxin.
1987
Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuteriated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuteriated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuteriated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3\u20134 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuteriated and all tryptophan aromatic protons were deuteriated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (
1983
Resonance Rayleigh light scattering was measured at the Soret absorption band of meso-tetraphenyl porphine (in the free-base form), chlorophyll a, and meso-tetra(4-pyridyl) porphine (in the fully prolonated form). The last compound, unlike the first two, showed unexpected fluorescence on the red side of, but close to, its Soret absorption band, and scattering experiments were not possible in the spectral range where fluorescence was detectable. The depolarization ratios measured for the Rayleigh scattered light of all three compounds were close to the value 1 8 expected for a planar oscillator, in line with previous conclusions that even when the in-plane degeneracy is lifted, the splitting of the in-plane bands at the Soret frequencies is small and the two transitions overlap considerably. Very good agreement was found between the measured scattering intensities and those predicted theoretically at the red edge of the Soret bands. Good agreement was also observed in the blue part of the Soret band in the case where measurements were possible (meso-tetra(4-pyridyl) porphine). However, in the intermediate spectral range the measured intensities exceeded appreciably the theoretically expected ones. It may thus be concluded that the enhanced scattering intensity that had been observed before in the red part of the last electronic absorption band of many compounds is not limited to such bands. The observed discrepancy between the experimentally determined intensities and those calculated from the absorption spectra may be due to momentary heterogeneity in the absorption coefficients, ???, in the population of absorbing molecules, the absorption and scattering depending each on different averages of ???. This heterogeneity is accentuated in the red part of the bands, where transitions from thermally excited molecules are more pronounced than in other parts of the absorption bands.
Every process of light absorption is inherently associated with a resonance scattering of the incident beam regardless of the nature of the absorbing molecules. Both the real and imaginary parts of the refractive index contribute to the resonance scattering, and marked depolarization is expected to be observed as a rule in the light scattered by complex molecules. The resonance and near-resonance Rayleigh scattering has been measured for a number of cyanine dyes: pseudocyanine, orthochrome T, 1,1′-diethyl-2, 2′-pyridyl- quinolyl cyanine, and pinacyanol chloride, in a number of solvents. In no case was the measured scattering intensity weaker than that expected theoretically, and good agreement between theory and experiment was found as a rule at the blue part of the absorption bands. In contrast, the measured scattering intensities exceeded (by up to fourfold) the theoretically expected values at the red parts of the absorption bands. Possible reasons for this discrepancy are discussed.
1981
Measurements of the depolarization ratio ρv of light scattered by the pigments lycopene and β-carotene at the red part of their absorption bands yielded values which are very close to the theoretical value 1/3 of a fully anisotropic molecular polarizability, i.e., that due to an electric dipole moment. Measurements of ρv at the blue edge of the visible absorption band of pinacyanol chloride yielded a value of 0.75 at 472.2 nm, which is the maximum value that a depolarization ratio can assume, and is attained if the average molecular polarizability is zero. This is possible only if the diagonalized polarizability tensor has at least one negative element to counterbalance the positive ones. A negative refractive index at the blue edge of the absorption band is thus experimentally demonstrated.
1979
The depolarized scattering intensity from the polyene pigment lycopene was measured in the visible absorptiion band and was found to be in good agreement with theoretical calculations at the short wavelength part of the band whereas significant deviations were observed at the red edge of this band.