Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.     

Regulation of Bin1 SH3 domain binding by phosphoinositides

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.     

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.     

Functional interaction between the SH2 domain of Fyn and tyrosine 324 of hamster polyomavirus middle-T antigen.

Middle-T antigen of mouse polyomavirus (MomT) associates with the cellular tyrosine kinases c-Src, c-Yes, and Fyn, while middle-T antigen of hamster polyomavirus (HamT) exclusively binds Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. Here we show that the kinase domain of Fyn is sufficient for association with MomT but not for binding of HamT. We further demonstrate that a Fyn mutant lacking the SH2 domain is able to bind MomT but fails to associate with HamT, indicating that the SH2 domain of Fyn is essential for stable association with HamT. HamT, but not MomT, contains a tyrosine residue, Tyr-324, in the sequence context YEEI. Mutation of Tyr-324 to phenylalanine led to a drastic reduction of associated Fyn and abolished the oncogenicity of HamT. This suggests that Tyr-324 is the major phosphotyrosine residue mediating the binding of HamT to the SH2 domain of Fyn. These findings show that mouse and hamster polyomaviruses use different strategies to target Src-related tyrosine kinases.     

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.     

The Lck SH3 domain negatively regulates localization to lipid rafts through an interaction with c-Cbl.

Lck is a member of the Src family of protein-tyrosine kinases and is essential for T cell development and function. Lck is localized to the inner surface of the plasma membrane and partitions into lipid rafts via dual acylation on its N terminus. We have tested the role of Lck binding domains in regulating Lck localization to lipid rafts. A form of Lck containing a point mutation inactivating the SH3 domain (W97ALck) was preferentially localized to lipid rafts compared with wild type or SH2 domain-inactive (R154K) Lck when expressed in Lck-deficient J.CaM1 cells. W97ALck incorporated more of the radioiodinated version of palmitic acid, 16-[(125)I]iodohexadecanoic acid. Overexpression of c-Cbl, a ligand of the Lck SH3 domain, depleted Lck from lipid rafts in Jurkat cells. Additionally, Lck localization to lipid rafts was enhanced in c-Cbl-deficient T cells. The association of Lck with c-Cbl in vivo required a functional SH3 domain. These results suggest a model whereby the SH3 domain negatively regulates basal localization of Lck to lipid rafts via association with c-Cbl.     

Regulation and function of SKAP-55 non-canonical motif binding to the SH3c domain of adhesion and degranulation-promoting adaptor protein

The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.     

SH3-SH2 domain orientation in Src kinases: NMR studies of Fyn

The regulatory domains of Src family kinases SH3 and SH2 suppress Src activity when bound to the catalytic domain. Here, the isolated SH3-SH2 fragment from the Src family member Fyn (FynSH32) is studied by NMR. The properties of this fragment are expected to be similar to the domains in the active state, where they are dissociated from the catalytic domain. Crosscommunication between SH3 and SH2 of FynSH32, measured by chemical shift perturbation, was found to be small. Diffusion and alignment anisotropy measurements showed that SH3 and SH2 of peptide-bound FynSH32 are significantly coupled but still exhibit some interdomain flexibility. The observed average domain orientation indicates that a large SH3-SH2 domain closure is required to reach the inactive state. The implications of these results for Src regulation are discussed.     

Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

Background  

Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking.     

Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions

Computational design of surface charge-charge interactions has been demonstrated to be an effective way to increase both the thermostability and the stability of proteins. To test the robustness of this approach for proteins with predominantly beta-sheet secondary structure, the chicken isoform of the Fyn SH3 domain was used as a model system. Computational analysis of the optimal distribution of surface charges showed that the increase in favorable energy per substitution begins to level off at five substitutions; hence, the designed Fyn sequence contained four charge reversals at existing charged positions and one introduction of a new charge. Three additional variants were also constructed to explore stepwise contributions of these substitutions to Fyn stability. The thermodynamic stabilities of the variants were experimentally characterized using differential scanning calorimetry and far-UV circular dichroism spectroscopy and are in very good agreement with theoretical predictions from the model. The designed sequence was found to have increased the melting temperature, DeltaT (m) = 12.3 +/- 0.2 degrees C, and stability, DeltaDeltaG(25 degrees C) = 7.1 +/- 2.2 kJ/mol, relative to the wild-type protein. The experimental data suggest that a significant increase in stability can be achieved through a very small number of amino acid substitutions. Consistent with a number of recent studies, the presented results clearly argue for a seminal role of surface charge-charge interactions in determining protein stability and suggest that the optimization of surface interactions can be an attractive strategy to complement algorithms optimizing interactions in the protein core to further enhance protein stability.     

Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.     

Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation

SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.     

Generation,characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain

The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a K_D of 0.9 nM and k_off of 6.6x10⁻⁴ s⁻¹, and to selectively inhibit chymase activity with an IC₅₀ value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.     

Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis.     

Generation,characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain

The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a K_D of 0.9 nM and k_off of 6.6x10^?4 s^?1, and to selectively inhibit chymase activity with an IC₅₀ value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.     

Direct binding of Grb2 SH3 domain to FGFR2 regulates SHP2 function

The adaptor protein Grb2 is recruited to intracellular early signalling complexes of many receptor tyrosine kinases and plays an important role transducing signals leading to MAP kinase activation. To date the SH2 domain of Grb2 has been shown to mediate receptor interactions with phosphorylated tyrosine residues sited directly on the receptor or on auxiliary docking proteins. Here we report that FGFR2 recruits Grb2 through its C-terminal SH3 domain. The binding site of this domain was mapped to the proline-rich C-terminus of the receptor. Deletion of the last 10 amino acids of FGFR2 abrogates interaction with Grb2. Synthetic peptides based on the C-terminus of FGFR2 bind to full length Grb2 with low micromolar affinity. The function of this novel mode of Grb2 binding provides resistance to site-specific Shp2-mediated receptor dephosphorylation.     

Acan125 binding to the SH3 domain of acanthamoeba myosin-IC

The domain organization of Acanthamoeba myosin-I, an oligomodular motor protein, includes a potentially important protein interaction module that is mostly uncharacterized. The Src homology 3, SH3, domain of myosin-I binds Acan125, a protein containing at least two consensus ligand binding domains: C-terminal SH3 binding motifs (PXXP) and N-terminal leucine-rich repeats. We report the first affinities determined for an SH3 domain of any myosin, namely, K(d) = 7 microM for a 21-residue synthetic peptide based on the PXXP domain sequence and K(d) = 0.15 microM for the PXXP domain included in the C-terminus of Acan125. These values are consistent with affinities reported for peptides and proteins that associate with SH3. By deletional analysis we show that only the PXXP domain is required for Acan125 to interact with the SH3 domain of Acanthamoeba myosin-IC (AmyoC(SH3)). The synthetic peptide described above at a concentration near the K(d) for SH3 binding blocked the interaction between native AmyoC and Acan125, mapping the interaction to the PXXP domain of Acan125 and the SH3 domain of myosin-I. These results are consistent with prototypical SH3 binding and suggest that a PXXP module is both necessary and sufficient to interact with an SH3 module of myosin-I.     

Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

Fyn is a nonreceptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases. Fyn plays an important role in inflammatory processes and neuronal functions. To generate a synthetic affinity reagent that can be used to probe Fyn, a phage-display library of fibronectin type III monobodies was affinity selected with the Src Homology 3 (SH3) domain of Fyn and three binders were isolated. One of the three binders, G9, is specific in binding to the SH3 domain of Fyn, but not to the other members of the Src family (i.e. Blk, Fgr, Hck, Lck, Lyn, Src and Yes), even though they share 51-81% amino acid identity. The other two bind principally to the Fyn SH3 domain, with some cross-reactivity to the Yes SH3 domain. The G9 binder has a dissociation constant of 166±6nM, as measured by isothermal titration calorimetry, and binds only to the Fyn SH3 domain out of 150 human SH3 domains examined in an array. Interestingly, although the G9 monobody lacks proline in its randomized BC and FG loops, it binds at the same site on the SH3 domain as proline-rich ligands, as revealed by competition assays. The G9 monobody, identified in this study, may be used as a highly selective probe for detecting and purifying cellular Fyn kinase.     

Differential binding to and regulation of JAK2 by the SH2 domain and N-terminal region of SH2-bbeta

SH2-Bbeta has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bbeta (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bbeta and SH2-Bbeta(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bbeta binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in alphaSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bbeta coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. DeltaC555 (amino acids 1 to 555 of SH2-Bbeta) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bbeta constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bbeta to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bbeta is necessary and sufficient for the stimulatory effect of SH2-Bbeta on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, DeltaC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. DeltaC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bbeta has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bbeta around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.     

Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck: PPIIhGAP

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PP_II helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PP_II helices led us to investigate whether proximal placement of a PP_II helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PP_II helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Eschericheria coli. The fusion protein, SH3_Hck: PPII_hGAP, folded spontaneously into a structure in which the PP_II helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3_Hck: PPII_hGAP fusion protein is useful for investigating SH3: PP_II helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.