Mapping discontinuous protein‐binding sites via structure‐based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure‐based approach and an experimental, high‐throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein–protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well‐characterised murine IgG1 antibody HyHEL‐5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL‐5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

Competition between citrate and heptapeptide DDSDEEN binding to DNA in presence of divalent cations

The binding of citrate and acidic peptide DDSDEEN with DNA in the presence of divalent cations is compared. Citric acid shows a higher number of binding sites on the DNA compared to the peptide; this is probably due to the bigger sitric hindrance of the peptide compared to the citric acid for the binding in the DNA grooves. Moreover, DNA preincubated with saturating amounts of citric acid is not available for the binding with successively added peptide. Therefore the peptide and citrate binding sites to some extent overlap.     

Comparative interaction kinetics of two recombinant fabs and of the corresponding antibodies directed to the coat protein of tobacco mosaic virus

Two recombinant Fab fragments, 57P and 174P, recognizing peptide 134–146 of the coat protein of tobacco mosaic virus have been cloned, sequenced and expressed in Escherichia coli. They differ by 15 amino acid changes in the sequence of their variable region. The interaction kinetics of the Fabs with the wild-type and four mutant peptides have been compared using a BIAcore^TM biosensor instrument. The recombinant Fab 174P had the same reactivity as the Fab fragment obtained by enzymatic cleavage of monoclonal antibody 174P. The two recombinant Fabs recognized the various peptides in the same ranking order but Fab 174P consistently dissociated somewhat faster from the peptides compared to Fab 57P. The two whole antibodies showed the same relative differences in reactivity as the two recombinant Fabs. The location of amino acid changes was visualized on a model structure of the Fab. Differences in dissociation rates of the two antibodies are most likely due to changes located at the periphery of the antigen-combining site and/or at the interface between the light and heavy chain domains. Our results demonstrate the feasibility of detecting very small differences in binding affinity by the biosensor technology, which is a prerequisite for assessing the functional effect of limited structural changes.     

Using synthetic peptides and recombinant collagen to understand DDR–collagen interactions

The discoidin domain receptors, DDR1 and DDR2, are a subfamily of receptor tyrosine kinases that are activated upon binding to collagen. DDR–collagen interactions play an important role in cell proliferation and migration. Over the past few decades, synthetic peptides and recombinant collagen have been developed as tools to study the biophysical characteristics of collagen and various protein–collagen interactions. Herein we review how these techniques have been used to understand DDR–collagen interactions. Using synthetic collagen-like peptides, the GVM-GFO motif has been found to be the major binding site on collagens II and III for DDR1 and DDR2. An X-ray co-crystal structure of the DDR2 DS domain bound to a synthetic collagen-like peptide containing the GVM-GFO motif further provides molecular details of the DDR–collagen interactions. Recombinant collagen has also been used to provide further validation of the GVM-GFO binding motif. Although GVM-GFO has been defined as the minimal binding site, in synthetic peptide studies at least two triplets N-terminal to the essential GVM-GFO binding motif in collagen III sequence are needed for DDR2 activation at high peptide concentrations.     

Epitope mapping of the neuronal growth inhibitor Nogo-A for the Nogo receptor and the cognate monoclonal antibody IN-1 by means of the SPOT technique

Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15 mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 F(ab) fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x not equal to P), which occurs within the so-called Nogo66 region (residues 1054-1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 F(ab) fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 F(ab) fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334-966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 F(ab) fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive.     

Sub‐angstrom modeling of complexes between flexible peptides and globular proteins

A wide range of regulatory processes in the cell are mediated by flexible peptides that fold upon binding to globular proteins. Computational efforts to model these interactions are hindered by the large number of rotatable bonds in flexible peptides relative to typical ligand molecules, and the fact that different peptides assume different backbone conformations within the same binding site. In this study, we present Rosetta FlexPepDock, a novel tool for refining coarse peptide–protein models that allows significant changes in both peptide backbone and side chains. We obtain high resolution models, often of sub‐angstrom backbone quality, over an extensive and general benchmark that is based on a large nonredundant dataset of 89 peptide–protein interactions. Importantly, side chains of known binding motifs are modeled particularly well, typically with atomic accuracy. In addition, our protocol has improved modeling quality for the important application of cross docking to PDZ domains. We anticipate that the ability to create high resolution models for a wide range of peptide–protein complexes will have significant impact on structure‐based functional characterization, controlled manipulation of peptide interactions, and on peptide‐based drug design. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

A computational method for selecting short peptide sequences for inorganic material binding

Discovering or designing biofunctionalized materials with improved quality highly depends on the ability to manipulate and control the peptide‐inorganic interaction. Various peptides can be used as assemblers, synthesizers, and linkers in the material syntheses. In another context, specific and selective material‐binding peptides can be used as recognition blocks in mining applications. In this study, we propose a new in silico method to select short 4‐mer peptides with high affinity and selectivity for a given target material. This method is illustrated with the calcite (104) surface as an example, which has been experimentally validated. A calcite binding peptide can play an important role in our understanding of biomineralization. A practical aspect of calcite is a need for it to be selectively depressed in mining sites.     

Crystallographic and calorimetric analysis of peptide binding to OppA protein.

Isothermal titration calorimetry has been used to study the binding of 20 different peptides to the peptide binding protein OppA, and the crystal structures of the ligand complexes have been refined. This periplasmic binding protein, part of the oligopeptide permease system of Gram negative bacteria, has evolved to bind and enclose small peptides of widely varying sequences. The peptides used in this study have the sequence Lys-X-Lys, where X is any of the 20 commonly occurring amino acids. The various side-chains found at position 2 on the ligand fit into a hydrated pocket. The majority of side-chains are restrained to particular conformations within the pocket. Water molecules act as flexible adapters, matching the hydrogen-bonding requirements of the protein and ligand and shielding charges on the buried ligand. This use of water by OppA to broaden the repertoire of its binding site is not unique, but contrasts sharply with other proteins which use water to help bind ligands highly selectively. Predicting the thermodynamics of binding from the structure of the complexes is highly complicated by the influence of water on the system.     

Structural properties and peptide ligand binding of the capsid homology domains of human Arc

The activity-regulated cytoskeleton-associated protein (Arc) is important for synaptic plasticity and the normal function of the brain. Arc interacts with neuronal postsynaptic proteins, but the mechanistic details of its function have not been fully established. The C-terminal domain of Arc consists of tandem domains, termed the N- and C-lobe. The N-lobe harbours a peptide binding site, able to bind multiple targets. By measuring the affinity of human Arc towards various peptides from stargazin and guanylate kinase-associated protein (GKAP), we have refined its specificity determinants. We found two sites in the GKAP repeat region that bind to Arc and confirmed these interactions by X-ray crystallography. Phosphorylation of the stargazin peptide did not affect binding affinity but caused changes in thermodynamic parameters. Comparison of the crystal structures of three high-resolution human Arc-peptide complexes identifies three conserved C–H…π interactions at the binding cavity, explaining the sequence specificity of short linear motif binding by Arc. We further characterise central residues of the Arc lobe fold, show the effects of peptide binding on protein dynamics, and identify acyl carrier proteins as structures similar to the Arc lobes. We hypothesise that Arc may affect protein-protein interactions and phase separation at the postsynaptic density, affecting protein turnover and re-modelling of the synapse. The present data on Arc structure and ligand binding will help in further deciphering these processes.     

DNABind: A hybrid algorithm for structure‐based prediction of DNA‐binding residues by combining machine learning‐ and template‐based approaches

Accurate prediction of DNA‐binding residues has become a problem of increasing importance in structural bioinformatics. Here, we presented DNABind, a novel hybrid algorithm for identifying these crucial residues by exploiting the complementarity between machine learning‐ and template‐based methods. Our machine learning‐based method was based on the probabilistic combination of a structure‐based and a sequence‐based predictor, both of which were implemented using support vector machines algorithms. The former included our well‐designed structural features, such as solvent accessibility, local geometry, topological features, and relative positions, which can effectively quantify the difference between DNA‐binding and nonbinding residues. The latter combined evolutionary conservation features with three other sequence attributes. Our template‐based method depended on structural alignment and utilized the template structure from known protein–DNA complexes to infer DNA‐binding residues. We showed that the template method had excellent performance when reliable templates were found for the query proteins but tended to be strongly influenced by the template quality as well as the conformational changes upon DNA binding. In contrast, the machine learning approach yielded better performance when high‐quality templates were not available (about 1/3 cases in our dataset) or the query protein was subject to intensive transformation changes upon DNA binding. Our extensive experiments indicated that the hybrid approach can distinctly improve the performance of the individual methods for both bound and unbound structures. DNABind also significantly outperformed the state‐of‐art algorithms by around 10% in terms of Matthews's correlation coefficient. The proposed methodology could also have wide application in various protein functional site annotations. DNABind is freely available at http://mleg.cse.sc.edu/DNABind/ . Proteins 2013; 81:1885–1899. © 2013 Wiley Periodicals, Inc.     

Proteinase 3 hydrolysis of peptides derived from human elastin exon 24

Summary. In normal and pathological tissues, elastin-derived peptides proceed of elastin degradation by polymorphonuclear leukocyte proteases: elastase, cathepsin G and proteinase 3. They were demonstrated to have a chemotactic activity, to promote cell proliferation and protease release, . . .. To be biologically active, their structures, which reflect elastase specificity, must adopt a β-turn conformation which accommodate to the cell surface-located elastin binding protein. In this study, we establish that human elastin exon 24-derived peptides containing at least two repeated VGVAPG sequences are hydrolyzed by the proteinase 3 (Pr3). As shown by mass spectrometry analyses, the demonstrated cleavage sites are in agreement with previously reported Pr3 substrate specificity and its lengthy substrate binding site. The characterization of the Pr3-generated products indicate that they contain at least one GXXPG sequence known to stimulate cellular effects after binding to the elastin receptor.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Predicting permanent and transient protein–protein interfaces

Protein–protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Molecular dynamics simulation study on the interaction of collagen‐like peptides with gelatinase‐A (MMP‐2)

Although several models have been proposed for the interaction of collagen with gelatinase‐A (matrix metalloproteinases‐2 (MMP‐2)), the extensive role of each domain of gelatinase A in hydrolyzing the collagens with and without interruptions is still elusive. Molecular docking, molecular dynamics (MD) simulation, normal mode analysis (NMA) and framework rigidity optimized dynamics algorithm (FRODAN) based analysis were carried out to understand the function of various domains of MMP‐2 upon interaction with collagen like peptides. The results reveal that the collagen binding domain (CBD) binds to the C‐terminal of collagen like peptide with interruption. CBD helps in unwinding the loosely packed interrupted region of triple helical structure to a greater extent. It can be possible to speculate that the role of hemopexin (HPX) domain is to prevent further unwinding of collagen like peptide by binding to the other end of the collagen like peptide. The catalytic (CAT) domain then reorients itself to interact with the part of the unwound region of collagen like peptide for further hydrolysis. In conclusion the CBD of MMP‐2 recognizes the collagen and aids in unwinding the collagen like peptide with interruptions, and the HPX domain of MMP‐2 binds to the other end of the collagen allowing CAT domain to access the cleavage site. This study provides a comprehensive understanding of the structural basis of collagenolysis by MMP‐2. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 779–794, 2014.     

Pinpointing the interaction site between semaphorin-3A and its inhibitory peptide

Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A–NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l -phenylalanine or photo-l -leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A–NRP-1 interaction.     

The amino acid sequence of an active site peptide from the H,K-ATPase of gastric mucosa

The gastric H,K-ATPase is an active transport protein that is responsible for the maintenance of a large pH gradient across the secretory canaliculus of the mammalian parietal cell. Acid secretion across these epithelial cell membranes is coupled to the potassium-stimulated hydrolysis of ATP catalyzed by H,K-ATPase, but the mechanism of coupling between ion transport and ATP hydrolysis is unknown. In order to investigate the enzymatic mechanism of this coupling, a peptide derived from the ATP binding site of H,K-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate (FITC), into the active site before trypsin digestion of the protein. The labeling of the enzyme by FITC was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. At 100% inhibition of activity, 3.5 +/- 1.6 nmol of FITC were incorporated per mg of protein. The amino acid sequence of the active site peptide is His-Val-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Gln-Leu-Ser-Ile-Arg, and FITC reacts with the lysine. This sequence is very similar to sequences of fluorescein-labeled peptides from the ATP binding sites of Na,K-ATPase and Ca2+-ATPase, and suggests that the active site structures of these ion transport ATPases are similar.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide binding by protein disulfide isomerase, a resident protein of the endoplasmic reticulum lumen

Previously we had demonstrated by photoaffinity labeling that a 57-kDa protein of the endoplasmic reticulum can bind and become covalently linked to glycosylatable photoreactive peptides containing the sequence-Asn-Xaa-Ser/Thr-. Subsequently, it was found that this protein, called glycosylation site-binding protein, was a multifunctional protein, i.e. it was identical to protein disulfide isomerase (PDI), the beta-subunit of prolyl hydroxylase and thyroid hormone-binding protein. In this study, the peptide specificity for binding to this 57-kDa protein, hereafter called PDI, has been investigated in more detail using photoaffinity probes. The results reveal that although N-glycosylation by oligosaccharyl transferase in the endoplasmic reticulum has an absolute requirement for an hydroxyamino acid in the third amino acid residue of the glycosylation site sequence, no such specificity is observed in the binding of such peptides to PDI. In addition to the lack of specificity for an hydroxyamino acid in the third residue position, no specificity was observed for the asparagine residue in the first position. Thus, binding is not restricted to peptides containing N-glycosylation sites. We have investigated the discrepancy between this apparent lack of sequence specificity and earlier results indicating that binding of peptides to PDI was specific for N-glycosylation site sequences. We now demonstrate that PDI in the lumen of microsomes is more efficiently labeled by peptides containing photoreactive-Asn-Xaa-Ser/Thr- sequences than by nonacceptor site sequences because the former become glycosylated. This increased labeling does not occur because the glycosylated form of the probes are preferentially recognized by PDI. Rather, it appears that increased polarity of the affinity probe after attachment of the oligosaccharide chain prevents its exit from the sealed microsomes, in effect concentrating it within the lumen of the microsome. These results, coupled with other studies on the multifunctional nature of PDI, suggest that the observed peptide binding may be a manifestation of the ability of PDI to recognize the backbone of polypeptides in the lumen of the endoplasmic reticulum.