Distinct interactions between ubiquitin and the SH3 domains involved in immune signaling

The SH3 domain is a versatile protein interaction motif that generally recognizes proline rich sequences (PRS). Recently, it has been shown that some SH3 domains in the endocytotic pathway can bind to ubiquitin. Moreover, Phe73 in the SH3 domain has been proposed to be an important determinant of the interaction, as the SH3 domains having Tyr73, either naturally or by mutation, failed to bind. Since SH3 domains are also important in immune receptor signaling, we investigated the interactions between immunologically relevant SH3 domains and ubiquitin. We observed that some of these SH3 domains can also bind to ubiquitin. Interestingly, we found that Nck2-SH3-3 bound to ubiquitin despite its Tyr at residue 73 (Tyr56 in our actual construct), but that CD2BP1-SH3 failed to bind, even though it has Phe at an equivalent position. Through detailed NMR binding studies on SH3 domains with Phes and Tyrs at the 73 position, we found that the two types of SH3 domains exhibit mechanistic differences in ubiquitin binding. We showed that the relative contribution of each binding sub-region in both SH3 domains and ubiquitin is quite different in the two binding modes. Such results raise the possibility that the mechanistic variety of these immunologically relevant SH3 domains might contribute to their functional diversity.     

In vitro evolution of recognition specificity mediated by SH3 domains reveals target recognition rules

We have designed a repertoire of 10(7) different SH3 domains by grafting the residues that are represented in the binding surfaces of natural SH3 domains onto the scaffold of the human Abl-SH3 domain. This phage-displayed library was screened by affinity selection for SH3 domains that bind to the synthetic peptides, APTYPPPLPP and LSSRPLPTLPSP, which are peptide ligands for the human Abl or Src SH3 domains, respectively. By characterizing the isolates, we have observed that as few as two or three amino acid substitutions lead to dramatic changes in recognition specificity. We propose that the ability to shift recognition specificity with a small number of amino acid replacements is an important evolutionary characteristic of protein binding modules. Furthermore, we have used the information obtained by these in vitro evolution experiments to generate a scoring matrix that evaluates the probability that any SH3 domain binds to the peptide ligands for the Abl and Src SH3 domains. A table of predictions for the 28 SH3 domains of baker's yeast is presented.     

Characterization of domain-peptide interaction interface: prediction of SH3 domain-mediated protein-protein interaction network in yeast by generic structure-based models

Determination of the binding specificity of SH3 domain, a peptide recognition module (PRM), is important to understand their biological functions and reconstruct the SH3-mediated protein-protein interaction network. In the present study, the SH3-peptide interactions for both class I and II SH3 domains were characterized by the intermolecular residue-residue interaction network. We developed generic MIEC-SVM models to infer SH3 domain-peptide recognition specificity that achieved satisfactory prediction accuracy. By investigating the domain-peptide recognition mechanisms at the residue level, we found that the class-I and class-II binding peptides have different binding modes even though they occupy the same binding site of SH3. Furthermore, we predicted the potential binding partners of SH3 domains in the yeast proteome and constructed the SH3-mediated protein-protein interaction network. Comparison with the experimentally determined interactions confirmed the effectiveness of our approach. This study showed that our sophisticated computational approach not only provides a powerful platform to decipher protein recognition code at the molecular level but also allows identification of peptide-mediated protein interactions at a proteomic scale. We believe that such an approach is general to be applicable to other domain-peptide interactions.     

Synthetic peptide arrays for investigating protein interaction domains

Synthetic peptide array technology was first developed in the early 1990s by Ronald Frank. Since then the technique has become a powerful tool for high throughput approaches in biology and biochemistry. Here, we focus on peptide arrays applied to investigate the binding specificity of protein interaction domains such as WW, SH3, and PDZ domains. We describe array-based methods used to reveal domain networks in yeast, and briefly review rules as well as ideas about the synthesis and application of peptide arrays. We also provide initial results of a study designed to investigate the nature and evolution of SH3 domain interaction networks in eukaryotes.     

Ubiquitin binds to and regulates a subset of SH3 domains

SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that bind to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain and that ubiquitin binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands.     

Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.

Many cellular signaling proteins contain SH3 (Src homology 3) domains that mediate protein interactions via specific proline-containing peptides. Unlike SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 binding site, the regulatory mechanism for SH3 interactions is unclear. p120 RasGAP (GTPase-activating protein), which contains an SH3 domain flanked by two SH2 domains, forms an abundant SH2-mediated complex with p190 RhoGAP in cells expressing activated tyrosine kinases. We have identified two closely linked tyrosine-containing peptides in p190 that bind simultaneously to the RasGAP SH2 domains upon p190 phosphorylation. This interaction is expected to bring the two SH2 domains into close proximity. Consequently, RasGAP undergoes a conformational change that results in a 100-fold increase in the accessibility of the target binding surface of its SH3 domain. These results indicate that the tandem arrangement of SH2 and SH3 domains found in a variety of cellular signaling proteins can provide a conformational mechanism for regulating SH3-dependent interactions through tyrosine phosphorylation. In addition, it appears that the role of p190 in the RasGAP signaling complex is to promote additional protein interactions with RasGAP via its SH3 domain.     

Architecture of basic building blocks in protein and domain structural interaction networks

MOTIVATION: The structural interaction of proteins and their domains in networks is one of the most basic molecular mechanisms for biological cells. Topological analysis of such networks can provide an understanding of and solutions for predicting properties of proteins and their evolution in terms of domains. A single paradigm for the analysis of interactions at different layers, such as domain and protein layers, is needed. RESULTS: Applying a colored vertex graph model, we integrated two basic interaction layers under a unified model: (1) structural domains and (2) their protein/complex networks. We identified four basic and distinct elements in the model that explains protein interactions at the domain level. We searched for motifs in the networks to detect their topological characteristics using a pruning strategy and a hash table for rapid detection. We obtained the following results: first, compared with a random distribution, a substantial part of the protein interactions could be explained by domain-level structural interaction information. Second, there were distinct kinds of protein interaction patterns classified by specific and distinguishable numbers of domains. The intermolecular domain interaction was the most dominant protein interaction pattern. Third, despite the coverage of the protein interaction information differing among species, the similarity of their networks indicated shared architectures of protein interaction network in living organisms. Remarkably, there were only a few basic architectures in the model (>10 for a 4-node network topology), and we propose that most biological combinations of domains into proteins and complexes can be explained by a small number of key topological motifs. CONTACT: doheon@kaist.ac.kr.     

SH3-SPOT: an algorithm to predict preferred ligands to different members of the SH3 gene family

We have developed a procedure to predict the peptide binding specificity of an SH3 domain from its sequence. The procedure utilizes information extracted from position-specific contacts derived from six SH3/peptide or SH3/protein complexes of known structure. The framework of SH3/peptide contacts defined on the structure of the complexes is used to build a residue-residue interaction database derived from ligands obtained by panning peptide libraries displayed on filamentous phage.The SH3-specific interaction database is a multidimensional array containing frequencies of position-specific contacts. As input, SH3-SPOT requires the sequence of an SH3 domain and of a query decapeptide ligand. The array, that we call the SH3-specific matrix, is then used to evaluate the probability that the peptide would bind the given SH3 domain. This procedure is fast enough to be applied to the entire protein sequence database.Panning experiments were performed to search putative specific ligands of different SH3 domains in a database of decapeptides, or in a database of protein sequences. The procedure ranked some of the natural partners of interaction of a number of SH3 domains among the best ligands of the approximately 5. 6x10(9) different decapeptides in the SWISSPROT database. We expect the predictive power of the method to increase with the enrichment of the SH3-specific matrix by interaction data derived from new complex structures or from the characterization of new ligands. The procedure was developed using the SH3 domain family as test case but its application can easily be extended to other families of protein domains (such as, SH2, MHC, EH, PDZ, etc.).     

Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.     

The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core

To investigate the relationships between sequence conservation, protein stability, and protein function, we have measured the thermodynamic stability, folding kinetics, and in vitro peptide-binding activity of a large number of single-site substitutions in the hydrophobic core of the Fyn SH3 domain. Comparison of these data to that derived from an analysis of a large alignment of SH3 domain sequences revealed a very good correlation between the distinct pattern of conservation observed at each core position and the thermodynamic stability of mutants. Conservation was also found to correlate well with the unfolding rates of mutants, but not to the folding rates, suggesting that evolution selects more strongly for optimal native state packing interactions than for maximal folding rates. Structural analysis suggests that residue-residue core packing interactions are very similar in all SH3 domains, which provides an explanation for the correlation between conservation and mutant stability effects studied in a single SH3 domain. We also demonstrate a correlation between stability and the in vivo activity of mutants, and between conservation and activity. However, the relationship between conservation and activity was very strong only for the three most conserved hydrophobic core positions. The weaker correlation between activity and conservation seen at the other seven core positions indicates that maintenance of protein stability is the dominant selective pressure at these positions. In general, the pattern of conservation at hydrophobic core positions appears to arise from conserved packing constraints, and can be effectively utilized to predict the destabilizing effects of amino acid substitutions.     

SH3 interactome conserves general function over specific form

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two‐hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain‐mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.     

Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.     

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15

Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.     

Promiscuous binding nature of SH3 domains to their target proteins

SH3 domains are small but important domains in cell-signaling and function through protein-protein interactions. Their promiscuous nature in binding to polyproline peptides makes them much more important because many SH3 domains from different proteins bind to different proteins having polyproline template on their surface. Very subtle changes in the sequence of SH3 domains and the binding peptides determine the specificity of the peptide binding. Recent observation that SH3 domains bind to non- proline peptides makes the scenario of peptide binding involving SH3 domains complicated. If domain swapped dimerization as observed in Eps8-SH3 domain also binds different peptides, it proves the versatility of the SH3 domains in binding to peptides in various ways. An overview of the promiscuity of SH3 domains has been discussed.     

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.