SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes. Significant diversity exists in protein structure and function, yet certain structural domains are used repeatedly across species to execute similar functions. The SH3 domain is one such common structural domain. It is found in signaling proteins and mediates protein–protein interactions by binding to short peptide sequences generally composed of proline. To investigate both the generality and selectivity of peptide binding by SH3 domains, we examined peptide specificity for almost all SH3 domains encoded within the proteome of the budding yeast, Saccharomyces cerevisiae, using a range of experimental methods. We found that although most of the intrinsic binding specificity for SH3 domains can be summarized by the two previously described canonical binding modes, each individual SH3 domain that we studied utilizes unique features of its cognate ligand to achieve binding selectivity. Moreover, some domains exhibit binding specificities that are distinct from the two canonical classes. We integrated peptide-SH3 domain binding data from three complementary screening techniques using a Bayesian statistical model to generate a protein–protein interaction network for the budding yeast SH3 domain family. This network was highly enriched in endocytosis proteins and their interactions. By examining these interactions in detail, we show that our SH3 domain network can be used to predict the temporal localization of several previously uncharacterized proteins to dynamic complexes that orchestrate the process of endocytosis.

Understanding the consequences on host physiology induced by viral infection requires complete understanding of the perturbations caused by virus proteins on the cellular protein interaction network. The VirusMINT database (http://mint.bio.uniroma2.it/virusmint/) aims at collecting all protein interactions between viral and human proteins reported in the literature. VirusMINT currently stores over 5000 interactions involving more than 490 unique viral proteins from more than 110 different viral strains. The whole data set can be easily queried through the search pages and the results can be displayed with a graphical viewer. The curation effort has focused on manuscripts reporting interactions between human proteins and proteins encoded by some of the most medically relevant viruses: papilloma viruses, human immunodeficiency virus 1, Epstein–Barr virus, hepatitis B virus, hepatitis C virus, herpes viruses and Simian virus 40.

A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 μM, while it contains as many as 50–80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment), that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are simultaneously analyzed in an array format, the likelihood of each interaction occurring in any given physiological settings can be evaluated. WISE can be easily extended to a variety of protein interaction domains, including those binding to modified peptides, thereby offering a powerful proteomic tool to help completing a full description of the cell interactome.

SH3 domains constitute a family of protein–protein interaction modules that bind to peptides displaying an X‐proline‐X‐X‐proline (XPXXP) consensus. We report that the SH3 domain of Eps8, a substrate of receptor and non‐receptor tyrosine kinases, displays a novel and unique binding preference. By a combination of approaches including (i) screening of phage‐displayed random peptide libraries, (ii) mapping of the binding regions on three physiological interactors of Eps8, (iii) alanine scanning of binding peptides and (iv) in vitro cross‐linking, we demonstrate that a proline‐X‐X‐aspartate‐tyrosine (PXXDY) consensus is indispensable for binding to the SH3 domain of Eps8. Screening of the Expressed Sequence Tags database allowed the identification of three Eps8‐related genes, whose SH3s also display unusual binding preferences and constitute a phylogenetically distinct subfamily within the SH3 family. Thus, Eps8 identifies a novel family of SH3‐containing proteins that do not bind to canonical XPXXP‐containing peptides, and that establish distinct interactions in the signaling network.