Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structure

Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.     

Ab initio prediction of peptide-MHC binding geometry for diverse class I MHC allotypes

Since determining the crystallographic structure of all peptide-MHC complexes is infeasible, an accurate prediction of the conformation is a critical computational problem. These models can be useful for determining binding energetics, predicting the structures of specific ternary complexes with T-cell receptors, and designing new molecules interacting with these complexes. The main difficulties are (1) adequate sampling of the large number of conformational degrees of freedom for the flexible peptide, (2) predicting subtle changes in the MHC interface geometry upon binding, and (3) building models for numerous MHC allotypes without known structures. Whereas previous studies have approached the sampling problem by dividing the conformational variables into different sets and predicting them separately, we have refined the Biased-Probability Monte Carlo docking protocol in internal coordinates to optimize a physical energy function for all peptide variables simultaneously. We also imitated the induced fit by docking into a more permissive smooth grid representation of the MHC followed by refinement and reranking using an all-atom MHC model. Our method was tested by a comparison of the results of cross-docking 14 peptides into HLA-A*0201 and 9 peptides into H-2K(b) as well as docking peptides into homology models for five different HLA allotypes with a comprehensive set of experimental structures. The surprisingly accurate prediction (0.75 A backbone RMSD) for cross-docking of a highly flexible decapeptide, dissimilar to the original bound peptide, as well as docking predictions using homology models for two allotypes with low average backbone RMSDs of less than 1.0 A illustrate the method's effectiveness. Finally, energy terms calculated using the predicted structures were combined with supervised learning on a large data set to classify peptides as either HLA-A*0201 binders or nonbinders. In contrast with sequence-based prediction methods, this model was also able to predict the binding affinity for peptides to a different MHC allotype (H-2K(b)), not used for training, with comparable prediction accuracy.     

Flexible docking of peptides to proteins using CABS‐dock

Molecular docking of peptides to proteins can be a useful tool in the exploration of the possible peptide binding sites and poses. CABS‐dock is a method for protein–peptide docking that features significant conformational flexibility of both the peptide and the protein molecules during the peptide search for a binding site. The CABS‐dock has been made available as a web server and a standalone package. The web server is an easy to use tool with a simple web interface. The standalone package is a command‐line program dedicated to professional users. It offers a number of advanced features, analysis tools and support for large‐sized systems. In this article, we outline the current status of the CABS‐dock method, its recent developments, applications, and challenges ahead.     

Characterization of prostate-specific antigen binding peptides selected by phage display technology

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.     

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.     

Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library.

Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg-Gly-Asp or Lys-Gly-Asp were discovered and synthesized. The cyclic, disulfide-bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds.     

Insight into the bovine milk peptide LPcin‐YK3 selection in the proteolytic system of Lactobacillus species

Antimicrobial peptides are class of small, positively charged peptides known for their broad‐spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin‐YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100‐fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide‐binding protein A and LPcin‐YK3 peptide. Given that degradation related to the LPcin‐YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin‐YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide‐binding protein A in respect of LPcin‐YK3 peptide.     

Phage display screening against a set of targets to establish peptide-based sugar mimetics and molecular docking to predict binding site

A novel selection approach is presented to screen phage display peptide libraries against sets of receptors that share specificity for the same ligand. This strategy was applied to the discovery of glycomimetic peptides. Through these screens, a number of peptide clones were discovered that bind the lectins used in the screen, in a sugar competitive manner. In addition, the majority of the selected peptides demonstrate sugar type mimicry consistent with lectin specificity. Docking studies were conducted to establish whether the mimetic peptides bind to the lectin ConA at the sugar binding site or to a nearby, alternative site shown to bind to YPY-containing peptides previously discovered from single-target screens. Of the three cyclic peptides subjected to computational docking, CNTPLTSRC had the highest predicted affinity and CSRILTAAC demonstrated specificity for the sugar binding site comparable to the natural ligand itself.     

Facile detection of specific RNA-polypeptide interactions by MALDI-TOF mass spectrometry.

A simple method for the detection of specific RNA-polypeptide interactions using MALDI-TOF mass spectroscopy is described. Instead of direct observation of the RNA-polypeptide complex, we attempted the indirect observation of the binding event by focusing on the disappearance of the free polypeptide signal upon interaction with RNA. As a result, specific binding of the Rev-response element (RRE) RNA of the HIV with two RRE-binding peptide aptamers, DLA and RLA peptides, as well as the bacteriophage lambda boxB RNA with the lambda N peptide was observed. We also show that specific RNA-binding peptides can be identified from a mixture of peptides with varying RNA-binding affinity, showing that the method could be applied to high-throughput screening from simple peptide libraries. The method described in this study provides a quick and simple method for detecting specific RNA-polypeptide interactions that avoids difficulties associated with direct observation of RNA and RNA-polypeptide complexes, which may find various applications in the analysis of RNA-polypeptide interactions and in the identification of novel RNA-binding polypeptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope

Human monoclonal antibody 2F5 is one of a few human antibodies that neutralize a broad range of HIV-1 primary isolates. The 2F5 epitope on gp41 includes the sequence ELDKWA, with the core residues, DKW, being critical for antibody binding. HIV-neutralizing antibodies have never been elicited by immunization with peptides bearing ELDKWA, suggesting that important part(s) of the 2F5 paratope remain unidentified. The use of longer peptides extending beyond ELDKWA has resulted in increased epitope antigenicity, but neutralizing antibodies have not been generated. We sought to develop peptides that bind to 2F5, and that function as specific probes of the 2F5 paratope. Thus, we used 2F5 to screen a set of phage-displayed, random peptide libraries. Tight-binding clones from the random peptide libraries displayed sequence variability in the regions flanking the DKW motif. To further reveal flanking regions involved in 2F5 binding, two semi-defined libraries were constructed having 12 variegated residues either N-terminal or C-terminal to the DKW core (X(12)-AADKW and AADKW-X(12), respectively). Three clones isolated from the AADKW-X(12) library had similar high affinities, despite a lack of sequence homology among them, or with gp41. The contribution of each residue of these clones to 2F5 binding was evaluated by Ala substitution and amino acid deletion studies, and revealed that each clone bound 2F5 by a different mechanism. These results suggest that the 2F5 paratope is formed by at least two functionally distinct regions: one that displays specificity for the DKW core epitope, and another that is multispecific for sequences C-terminal to the core epitope. The implications of this second, multispecific region of the 2F5 paratope for its unique biological function are discussed.     

A method for generation of arbitrary peptide libraries using genomic DNA

Random peptide libraries can be constructed either by in vitro synthesis of random peptides, or through translation of DNA sequences from synthetic random oligonucleotides. Here we describe an alternative way of making arbitrary peptide libraries with high diversity that can be used in screening as random peptide libraries. Genomic DNA digested with a frequent-cutting restriction enzyme recognizing four nucleotides will theoretically consist of small DNA pieces with average length of 256 nucleotides, and on average around 10⁷ fragments can be generated from a genome of 3 × 10⁹ bases. A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening experiments. Moreover, the same genome digested with a different four-cutter enzyme or ligated into different reading frames will result in different nonoverlapping libraries. A series of such libraries could be generated with genomic DNAs from different species. In this study, human genomic DNA was digested with four-cutter restriction enzymes DpnII and Tsp509I, respectively, and cloned into yeast expression vector pGADT7 to generate arbitrary peptide libraries. These libraries were used in yeast two-hybrid assays to screen for binding motifs of the PDZ domain containing protein synectin. Our results showed that in addition to various native carboxy-terminal tails, synectin could also bind to many artificial ones, some of which contained a consensus sequence—(S/T)XC-COOH.     

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.     

HER-2-based design of pertuzumab mimetic peptides

An in silico peptide design strategy is conducted in order to introduce a novel series of pertuzumab mimetic peptides, aimed to target the extracellular domain of HER-2 and prevent its signal transduction. A combination of alanine scanning and contact surface analysis is employed to assess the pertuzumab paratope, HER-2 epitope and their hot spots. Furthermore, the recognised residues are utilised to construct nine 10-mer peptides. Some of the peptides are modified pertuzumab paratope sequences, whereas the others are designed and modified as strongly binding complementary peptides for HER-2 epitope. Evaluation of the peptides is carried out through homology modelling, molecular dynamics simulation (MDS) and docking. It was revealed that all peptides mimic pertuzumab performance since they exhibit noticeable binding interaction energies, inhibitory efficiencies and number of hot spots. However, YNDSTHGERL with five hot spots and considerable interaction energy has the highest ability in interfering with HER-2-specific epitope. Inhibiting potency of this peptide is verified through MDSs.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

Interference of rheumatoid factor activity by aspartame,a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L ‐Asp‐L ‐Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo‐ and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. ( 1998 ). Clin. Pharmac. Ther. 63 , 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid‐phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl₂ to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG₁ antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer‐assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG₄ antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.     

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

Comprehensive analysis of the molecular docking of small molecule inhibitors to the Aβ1–40 peptide and its Osaka-mutant: insights into the molecular mechanisms of Aβ-peptide inhibition

Abstract

Alzheimer’s disease (AD) is the most common form of age-related neurodegeneration occurs because of deposition of proteins in the form of extracellular plaques containing aggregated amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles composed of aggregated microtubule-binding protein tau. Amyloid aggregation process can be enhanced by several familial AD-associated mutations in Aβ peptide. In this study, we have unravelled the interactions of 40 small molecule inhibitors with the Osaka-mutant of Aβ_1–40 peptide at atomic level and characterized modes of their binding to mutant Aβ by docking approaches. We have also compared docking energies of these inhibitors with Osaka-mutant with those previously determined for the wild-type and Iowa-mutant peptides and discussed in light of the peptide conformations and non-covalent interactions. We have also discussed inhibition mechanisms of these three peptides. Our analyses revealed that these small molecules can efficiently inhibit Osaka-mutant. The binding modes of drugs with these three peptides are markedly different and so are the mechanisms of inhibition of these three peptides. Overall analysis of the data reveals that binding energy of Iowa-mutant drug complex is lowest and most stable which is followed wild-type peptide-drug complex followed by Osaka-mutant drug complex.

Communicated by Ramaswamy H. Sarma     

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.     

DynaDock: A new molecular dynamics‐based algorithm for protein–peptide docking including receptor flexibility

Molecular docking programs play an important role in drug development and many well‐established methods exist. However, there are two situations for which the performance of most approaches is still not satisfactory, namely inclusion of receptor flexibility and docking of large, flexible ligands like peptides. In this publication a new approach is presented for docking peptides into flexible receptors. For this purpose a two step procedure was developed: first, the protein–peptide conformational space is scanned and approximate ligand poses are identified and second, the identified ligand poses are refined by a new molecular dynamics‐based method, optimized potential molecular dynamics (OPMD). The OPMD approach uses soft‐core potentials for the protein–peptide interactions and applies a new optimization scheme to the soft‐core potential. Comparison with refinement results obtained by conventional molecular dynamics and a soft‐core scaling approach shows significant improvements in the sampling capability for the OPMD method. Thus, the number of starting poses needed for successful refinement is much lower than for the other methods. The algorithm was evaluated on 15 protein–peptide complexes with 2–16mer peptides. Docking poses with peptide RMSD values <2.10 Å from the equilibrated experimental structures were obtained in all cases. For four systems docking into the unbound receptor structures was performed, leading to peptide RMSD values <2.12 Å. Using a specifically fitted scoring function in 11 of 15 cases the best scoring poses featured a peptide RMSD ≤2.10 Å. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.