Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-gamma1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.     

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.     

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.     

Crystal structure of the C-terminal SH2 domain of the p85alpha regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate.

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 A resolution of the C-terminal SH2 domain (C-SH2) of the P85alpha regulatory subunit of phosphoinositide 3-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (alphaA2) and Arg36 (betaB5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal.     

Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities

The yeast Fus1p SH3 domain binds to peptides containing the consensus motif, R(S/T)(S/T)SL, which is a sharp contrast to most SH3 domains, which bind to PXXP-containing peptides. Here, we have demonstrated that this domain binds to R(S/T)(S/T)SL-containing peptides derived from two putative in vivo binding partners from yeast proteins, Bnr1p and Ste5p, with K_d values in the low micromolar range. The R(S/T)(S/T)SL consensus motif is necessary, but not sufficient for binding to the Fus1p SH3 domain, as residues lying N-terminal to the consensus motif also play a critical role in the binding reaction. Through mutagenesis studies and comparisons to other SH3 domains, we have discovered that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface, which plays the predominant role in binding for most SH3 domains, is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, we created a version of the Fus1p SH3 domain that binds to a PXXP-containing peptide with extremely high affinity (K_d =  40 nM). Based on our data and analysis, we have clearly delineated two distinct surfaces comprising the typical SH3-domain-binding interface and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3-domain-mediated interaction described in the literature. Within this framework, dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of these two surfaces.     

Allosteric loss-of-function mutations in HIV-1 Nef from a long-term non-progressor

Activation of Src family kinases by human immunodeficiency virus type 1 (HIV-1) Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site influence Nef interactions allosterically with a key effector protein linked to AIDS progression.     

SH3 domain of Bruton's tyrosine kinase can bind to proline-rich peptides of TH domain of the kinase and p120cbl

X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B-cell differentiation; this incapacitates antibody production in XLA patients, who suffer from recurrent, sometimes lethal, bacterial infections. BTK plays an important role in B-cell development; it interacts with several proteins in the context of signal transduction. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in a patient family. To understand the role of BTK, we studied binding of BTK SH3 domain (aa 216–273, 58 residues) and truncated SH3 domain (216–259, 44 residues) with proline-rich peptides; the first peptide constitutes the SH3 domain of BTK, while the latter peptide lacks 14 amino acid residues of the C terminal. Proline-rich peptides selected from TH domain of BTK and p120^cbl were studied. It is known that BTK TH domain binds to SH3 domains of various proteins. We found that BTK SH3 domain binds to peptides of BTK TH domain. This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand-binding modules. We also found that truncated SH3 domain binds to BTK TH domain peptide less avidly than does normal SH3 domain. Also, we show that the SH3 and truncated SH3 domains bind to peptide of p120^cbl, but the latter domain binds weakly. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context, hence the weaker binding. These results delineate the importance of C terminal in binding of SH3 domains and indicate also that improper folding and the altered binding behavior of mutant BTK SH3 domain likely leads to XLA. Proteins 29:545–552, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain

Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was used to assess the performances of different kernel functions in SVM and different combinations of the MIECs. The best SVM classifier gave satisfactory predictions for the test set, indicated by average prediction accuracy rates of 78% and 91% for the binding and non-binding peptides, respectively. We also showed that the performance of our approach on both binding affinity prediction and binder/non-binder classification was superior to the performances of the conventional MM/Poisson-Boltzmann solvent-accessible surface area and MM/generalized Born solvent-accessible surface area calculations. Our study demonstrates that the analysis of the MIECs between peptides and the SH3 domain can successfully characterize the binding interface, and it provides a framework to derive integrated prediction models for different domain-peptide systems.     

Interaction between Btk TH and SH3 domain

Several mechanisms are involved in the regulation of cellular signaling. Bruton tyrosine kinase (Btk) of the Tec family contains in the Tec homology (TH) domain a proline-rich region (PRR) capable of interacting with several SH3 domains. The Btk has the SH3 domain adjacent to the TH domain. CD and fluorescence spectroscopy were used to study the binding of two peptides corresponding to segments in the PRR to the Btk SH3 domain. The peptide for the N-terminal half of the PRR binds specifically, whereas the other peptide had hardly any affinity. The TH domain has about four times lower affinity to the SH3 domain than the peptide, 17.0 vs 3.9 microM. The interaction was further tested with an SH3 domain construct that contained the PRR. The two peptides cannot compete for the binding to the extended protein and the TH domain has two times lower affinity to the extended SH3 domain. The intra- or intermolecular interaction between the TH and SH3 domain might have regulatory function also in the other Tec family members.     

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.     

Structural insight into the binding diversity between the human Nck2 SH3 domains and proline-rich proteins

Human Nck2 (hNck2) is a 380-residue adapter protein consisting of three SH3 domains and one SH2 domain. Nck2 plays a pivotal role in connecting and integrating signaling networks constituted by transmembrane receptors such as ephrinB and effectors critical for cytoskeletonal dynamics and remodeling. In this study, we aimed to determine the NMR structures and dynamic properties of the hNck2 SH3 domains and to define their ligand binding preferences with nine proline-rich peptides derived from Wire, CAP-1, CAP-2, Prk, Wrch1, Wrch2, and Nogo. The results indicate (1) the first hNck2 SH3 domain is totally insoluble. On the other hand, although the second and third hNck2 SH3 domains adopt a conserved SH3 fold, they exhibit distinctive dynamic properties. Interestingly, the third SH3 domain has a far-UV CD spectrum typical of a largely unstructured protein but exhibits {1H}-15N steady-state NOE values larger than 0.7 for most residues. (2) The HSQC titrations revealed that the two SH3 domains have differential ligand preferences. The second SH3 domain seems to prefer a consensus sequence of APx#PxR, while the third SH3 domain prefers PxAPxR. (3) Several high-affinity bindings were identified for hNck2 SH3 domains by isothermal titration calorimetry. In particular, the binding of SH3-3 with the Nogo-A peptide was discovered and shown to exhibit a Kd of 5.7 microM. Interestingly, of the three SH3-binding motifs carried by Wrch1, only the middle one was capable of binding SH3-2. Our results provide valuable clues for further functional investigations into the Nck2-mediated signaling networks.     

Binding, domain orientation, and dynamics of the Lck SH3-SH2 domain pair and comparison with other Src-family kinases

The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.     

Prediction of binding affinities between the human amphiphysin-1 SH3 domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis

The SH3 domain of the human protein amphiphysin-1, which plays important roles in clathrin-mediated endocytosis, actin function and signaling transduction, can recognize peptide motif PXRPXR (X is any amino acid) with high affinity and specificity. We have constructed a complex structure of the amphiphysin-1 SH3 domain and a high-affinity peptide ligand PLPRRPPRA using homology modeling and molecular docking, which was optimized by molecular dynamics (MD). Three-dimensional quantitative structure-affinity relationship (3D-QSAR) analyses on the 200 peptides with known binding affinities to the amphiphysin-1 SH3 domain was then performed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The best CoMSIA model showed promising predictive power, giving good predictions for about 95% of the peptides in the test set (absolute prediction errors less than 1.0). It was used to validate peptide-SH3 binding structure and provide insight into the structural requirements for binding of peptides to SH3 domains. Finally, MD simulations were performed to analyze the interaction between the SH3 domain and another peptide GFPRRPPPRG that contains with the PXRPXsR (s represents residues with small side chains) motif. MD simulations demonstrated that the binding conformation of GFPRRPPPRG is quite different from that of PLPRRPPRAA especially the four residues at the C terminal, which may explain why the CoMSIA model cannot give good predictions on the peptides of the PXRPXsR motif. Because of its efficiency and predictive power, the 3D-QSAR model can be used as a scoring filter for predicting peptide sequences bound to SH3 domains.     

Determinants of intra versus intermolecular self-association within the regulatory domains of Rlk and Itk

A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveal that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveal that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, we varied the length of the linker in both Rlk and Itk and analyzed the resulting variants by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins.     

The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity.

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.     

Structural basis of PxxDY motif recognition in SH3 binding

We have determined the solution structure of epidermal growth factor receptor pathway substrate 8 (Eps8) L1 Src homology 3 (SH3) domain in complex with the PPVPNPDYEPIR peptide from the CD3ε cytoplasmic tail. Our structure reveals the distinct structural features that account for the unusual specificity of the Eps8 family SH3 domains for ligands containing a PxxDY motif instead of canonical PxxP ligands. The CD3ε peptide binds Eps8L1 SH3 in a class II orientation, but neither adopts a polyproline II helical conformation nor engages the first proline-binding pocket of the SH3 ligand binding interface. Ile531 of Eps8L1 SH3, instead of Tyr or Phe residues typically found in this position in SH3 domains, renders this hydrophobic pocket smaller and nonoptimal for binding to conventional PxxP peptides. A positively charged arginine at position 512 in the n-Src loop of Eps8L1 SH3 plays a key role in PxxDY motif recognition by forming a salt bridge to D7 of the CD3ε peptide. In addition, our structural model suggests a hydrogen bond between the hydroxyl group of the aromatic ring of Y8 and the carboxyl group of E496, thus explaining the critical role of the PxxDY motif tyrosine residue in binding to Eps8 family SH3. These finding have direct implications also for understanding the atypical binding specificity of the amino-terminal SH3 of the Nck family proteins.     

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the α-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the “unbound” and “bound” states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2∼P7∼P10 > P9∼P6 > P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.