PeptideMine - A webserver for the design of peptides for protein-peptide binding studies derived from protein-protein interactomes

Background: Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues. Results: Here we describe an integrated approach called ``PeptideMine'' for the identification of peptides based on specific functional patterns present in the sequence of an interacting protein. This approach based on sequence searches in the interacting sequence space has been developed into a webserver, which can be used for the identification and analysis of peptides, peptide homologues or functional patterns from the interacting sequence space of a protein. To further facilitate experimental validation, the PeptideMine webserver also provides a list of physico-chemical parameters corresponding to the peptide to determine the feasibility of using the peptide for in vitro biochemical or biophysical studies. Conclusions: The strategy described here involves the integration of data and tools to identify potential interacting partners for a protein and design criteria for peptides based on desired biochemical properties. Alongside the search for interacting protein sequences using three different search programs, the server also provides the biochemical characteristics of candidate peptides to prune peptide sequences based on features that are most suited for a given experiment. The PeptideMine server is available at the URL: http://caps.ncbs.res.in/peptidemine

An automated framework for understanding structural variations in the binding grooves of MHC class II molecules

Background: MHC/HLA class II molecules are important components of the immune system and play a critical role in processes such as phagocytosis. Understanding peptide recognition properties of the hundreds of MHC class II alleles is essential to appreciate determinants of antigenicity and ultimately to predict epitopes. While there are several methods for epitope prediction, each differing in their success rates, there are no reports so far in the literature to systematically characterize the binding sites at the structural level and infer recognition profiles from them. Results: Here we report a new approach to compare the binding sites of MHC class II molecules using their three dimensional structures. We use a specifically tuned version of our recent algorithm, PocketMatch. We show that our methodology is useful for classification of MHC class II molecules based on similarities or differences among their binding sites. A new module has been used to define binding sites in MHC molecules. Comparison of binding sites of 103 MHC molecules, both at the whole groove and individual sub-pocket levels has been carried out, and their clustering patterns analyzed. While clusters largely agree with serotypic classification, deviations from it and several new insights are obtained from our study. We also present how differences in sub-pockets of molecules associated with a pair of autoimmune diseases, narcolepsy and rheumatoid arthritis, were captured by PocketMatch(13). Conclusion: The systematic framework for understanding structuralvariations in MHC class II molecules enables large scale comparison of binding grooves and sub-pockets, which is likely to have direct implications towards predicting epitopes and understanding peptide binding preferences.

An automated framework for understanding structural variations in the binding grooves of MHC class II molecules

Background: MHC/HLA class II molecules are important components of the immune system and play a critical role in processes such as phagocytosis. Understanding peptide recognition properties of the hundreds of MHC class II alleles is essential to appreciate determinants of antigenicity and ultimately to predict epitopes. While there are several methods for epitope prediction, each differing in their success rates, there are no reports so far in the literature to systematically characterize the binding sites at the structural level and infer recognition profiles from them. Results: Here we report a new approach to compare the binding sites of MHC class II molecules using their three dimensional structures. We use a specifically tuned version of our recent algorithm, PocketMatch. We show that our methodology is useful for classification of MHC class II molecules based on similarities or differences among their binding sites. A new module has been used to define binding sites in MHC molecules. Comparison of binding sites of 103 MHC molecules, both at the whole groove and individual sub-pocket levels has been carried out, and their clustering patterns analyzed. While clusters largely agree with serotypic classification, deviations from it and several new insights are obtained from our study. We also present how differences in sub-pockets of molecules associated with a pair of autoimmune diseases, narcolepsy and rheumatoid arthritis, were captured by PocketMatch(13). Conclusion: The systematic framework for understanding structural variations in MHC class II molecules enables large scale comparison of binding grooves and sub-pockets, which is likely to have direct implications towards predicting epitopes and understanding peptide binding preferences.

Deciphering complex patterns of class-I HLA-peptide cross-reactivity via hierarchical grouping

T-cell responses in humans are initiated by the binding of a peptide antigen to a human leukocyte antigen (HLA) molecule. The peptide-HLA complex then recruits an appropriate T cell, leading to cell-mediated immunity. More than 2000 HLA class-I alleles are known in humans, and they vary only in their peptide-binding grooves. The polymorphism they exhibit enables them to bind a wide range of peptide antigens from diverse sources. HLA molecules and peptides present a complex molecular recognition pattern, as many peptides bind to a given allele and a given peptide can be recognized by many alleles. A powerful grouping scheme that not only provides an insightful classification, but is also capable of dissecting the physicochemical basis of recognition specificity is necessary to address this complexity. We present a hierarchical classification of 2010 class-I alleles by using a systematic divisive clustering method. All-pair distances of alleles were obtained by comparing binding pockets in the structural models. By varying the similarity thresholds, a multilevel classification was obtained, with 7 supergroups, each further subclassifying to yield 72 groups. An independent clustering performed based only on similarities in their epitope pools correlated highly with pocket-based clustering. Physicochemical feature combinations that best explain the basis of clustering are identified. Mutual information calculated for the set of peptide ligands enables identification of binding site residues contributing to peptide specificity. The grouping of HLA molecules achieved here will be useful for rational vaccine design, understanding disease susceptibilities and predicting risk of organ transplants.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Structures of new crystal forms of Mycobacterium tuberculosis peptidyl-tRNA hydrolase and functionally important plasticity of the molecule

The X-ray structures of new crystal forms of peptidyl-tRNA hydrolase from M.similar to tuberculosis reported here and the results of previous X-ray studies of the enzyme from different sources provide a picture of the functionally relevant plasticity of the protein molecule. The new X-ray results confirm the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding and tRNA-binding sites. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movements is not, however, observed in the NMR structure.

Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation of E. coli Maltose Binding Protein

SecB, a soluble cytosolic chaperone component of the Secexport pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (ΔTm =7( 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability ofMBP(ΔTm=1(0.4 K) or preMBP (ΔTm=0(0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils.Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can reatly influence protein stability and aggregation propensity.

First Structural View of a Peptide Interacting with the Nucleotide Binding Domain of Heat Shock Protein 90

The involvement of Hsp90 in progression of diseases like cancer, neurological disorders and several pathogen related conditions is well established. Hsp90, therefore, has emerged as an attractive drug target for many of these diseases. Several small molecule inhibitors of Hsp90, such as geldanamycin derivatives, that display antitumor activity, have been developed and are under clinical trials. However, none of these tested inhibitors or drugs are peptide-based compounds. Here we report the first crystal structure of a peptide bound at the ATP binding site of the N-terminal domain of Hsp90. The peptide makes several specific interactions with the binding site residues, which are comparable to those made by the nucleotide and geldanamycin. A modified peptide was designed based on these interactions. Inhibition of ATPase activity of Hsp90 was observed in the presence of the modified peptide. This study provides an alternative approach and a lead peptide molecule for the rational design of effective inhibitors of Hsp90 function.

Bilirubin binding to polypeptides and chiral amines. Induced circular dichroism and fluorescence studies

Induced Cotton effects have been observed in the visible region on interaction of bilirubin with chiral mono- and diamines and poly-l-lysine. At alkaline pH distinct CD spectra are observed for bilirubin bound to the α-helical and β-sheet conformation of poly-l-lysine, which differ from that observed for the pigment bound to human serum albumin. The CD pattern observed on binding to N-acetyl-Lys-N1-methylamide in CH2Cl2 and dioxane is different from that observed in the presence of l-Ala-NH-(CH2)6-NH-l-Ala in dioxane. The latter case resembles the spectrum observed in the presence of human serum albumin. Binding to the helical polypeptide melittin and the antiparallel β-sheet peptide, gramicidin S, in aqueous solutions results in opposite signs of the bilirubin CD bands. The quenching of tryptophan fluorescence in melittin, in aqueous solution and enhancement of bilirubin fluorescence in dioxane on binding to gramicidin S have been used to monitor pigment-peptide interactions. The results suggest the utility of bilirubin as a conformational probe.

Fluorescence And Nmr-Studies Of The Binding Of Cholinergic Fluorescent-Probes To Horse Serum-Cholinesterase

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Theoretical studies on penicillins, penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues: binding specificities of transeptidases and penicillinases

Empirical potential energy calculations have been carried out to determine the preferred conformations of penicillins and penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues. With the exception of 1-oxa-1-dethia penicillins, all the other compounds favour C2 and the C3 puckered conformations of their five-membered rings. Replacement of C2 methyl groups by hydrogen atoms as in bisnorpenicillin V or oxidation of sulphur in position 1 as in sulphones, makes the C3 puckered form much less favourable. Addition of an amino-acyl group at the C6 atom, however, makes the C3 puckered form more favoured in penicillin G or V and in 1-carba-1-dethia penicillins. Through the replacement of the sulphur atom at position 1 by an oxygen atom or by a -CH2 group increases the non-planarity of the lactam peptide bond, it significantly affects the relative disposition of the C3 carboxyl group with respect to the β-lactam ring. These conformational differences have been correlated with the biological activities of these compounds. The present study suggests that the conformation of the bicyclic ring system may be more important for initial binding with the crosslinking enzyme(s) involved in the biosynthesis of bacterial cell-wall peptidoglycan and that the mode of binding is influenced by the nature of the side-group at the C6 atom. These studies predict, in agreement with experimental results, that the 1-oxa-1-dethia penicillin nulceus is an inhibitor of penicillianses. The study also suggests that the stereospecificities of the crosslinking enzyme(s) and penicillinases are very similar with regard to the nature of the side-group at the 6 atom and the confirmation of the bicyclic ring system. However, the confirmational requirement for the bicyclic ring system appears to be more specific in the former enzyme than in the latter.

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 Å resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is dose to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.