The SH3 domain of nebulin binds selectively to type II peptides: theoretical prediction and experimental validation

Nebulin, a giant modular protein from muscle, is thought to act as a molecular ruler in sarcomere assembly. The C terminus of nebulin, located in the sarcomere Z-disk, comprises an SH3 domain, a module well known for its role in protein/protein interactions. SH3 domains are known to recognize proline-rich ligands, which have been classified as type I or type II, depending on their relative orientation with respect to the SH3 domain in the complex formed. Type I ligands are bound with their N terminus at the RT loop of the SH3 domain, while type II ligands are bound with their C terminus at the RT loop. Many SH3 domains can bind peptides of either class. Despite the potential importance of the SH3 domain for the function of nebulin as an integral part of a complex network of interactions, no in vivo partner has been identified so far. We have adopted an integrated approach, which combines bioinformatic tools with experimental validation to identify possible partners of nebulin SH3. Using the program SPOT, we performed an exhaustive screening of the muscle sequence databases. This search identified a number of potential nebulin SH3 partners, which were then tested experimentally for their binding affinity. Synthetic peptides were studied by both fluorescence and NMR spectroscopy. Our results show that nebulin SH3 domain binds selectively to type II peptides. The affinity for a type II peptide, 12 residues long, spanning the sequence of a stretch of titin known to colocalise with nebulin in the Z-disk is in the submicromolar range (0.7 microM). This affinity is among the highest found for SH3/peptide complexes, suggesting that the identified stretch could have significance in vivo. The strategy outlined here is of more general applicability and may provide a valuable tool to identify potential partners of SH3 domains and of other peptide-binding modules.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

快速构建蛋白质结构域克隆库的方法

对蛋白质组学的研究有许多不同的切入方法 .从研究的生物学意义和可行性考虑 ,提出从蛋白结构域入手进行蛋白质组学研究 .SH2 (Srchomology 2 )结构域是细胞信号转导中重要的元件之一 ,人SH2结构域共有约 12 0种 ,对其进行研究将深刻揭示细胞信号转导的规律 .为了得到人所有的SH2结构域序列及克隆 ,首先在公共数据库里检索出了人所有的SH2结构域序列 ,利用国际上现有的共享资源IMAGE(IntegratedMolecularAnalysisofGenomesandTheirExpression)克隆为PCR模板 ,解决了从cDNA文库中难以克隆低丰度结构域的问题 .利用有方向性的TOPO克隆技术提高克隆效率 ,从而快速高效地构建了包括 6 0个SH2结构域的克隆库 .克隆库可以方便地转换到GATEWAY系统具有各种用途的载体上 ,为SH2结构域的蛋白质组学研究奠定了坚实的基础     

Identification of p115 as a PLCgamma1-binding protein and the role of Src homology domains of PLCgamma1 in the vesicular transport

In order to gain further insight into the function(s) of PLCgamma1, we tried to identify the binding partners that can interact with the SH223 domains of PLCgamma1. Immunoscreening was performed with the purified antisera that are specific to SH223-binding proteins. Several immunoreactive clones were identified as the putative binding proteins and one of them was identified as p115. p115 was reported to be required for transcytotic fusion and subsequent binding of the vesicles to the target membrane. The interaction between PLCgamma1 and p115 was specific to carboxyl-terminal SH2 domain and SH3 domain of PLCgamma1, and also confirmed by biochemical approaches such as co-immunoprecipitation, pull-down assay, and glycerol gradient fractionation. To further characterize the role of SH domains of PLCgamma1 in the vesicle transport pathway, secreted form of alkaline phosphatase (SEAP) reporter assay was carried out. When the SH2 and/or SH3 domains of PLCgamma1 were deleted, the secretion of SEAP was significantly reduced. These findings indicate that the SH2 and SH3 domains of PLCgamma1 may play a role(s) in the process of the vesicle transport via interaction with other vesicle-associated proteins such as p115.     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

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.     

A lack of peptide binding and decreased thermostability suggests that the CASKIN2 scaffolding protein SH3 domain may be vestigial

Background

CASKIN2 is a neuronal signaling scaffolding protein comprised of multiple ankyrin repeats, two SAM domains, and one SH3 domain. The CASKIN2 SH3 domain for an NMR structural determination because its peptide-binding cleft appeared to deviate from the repertoire of aromatic enriched amino acids that typically bind polyproline-rich sequences.

Results

The structure demonstrated that two non-canonical basic amino acids (K290/R319) in the binding cleft were accommodated well in the SH3 fold. An K290Y/R319W double mutant restoring the typical aromatic amino acids found in the binding cleft resulted in a 20 °C relative increase in the thermal stability. Considering the reduced stability, we speculated that the CASKIN2 SH3 could be a nonfunctional remnant in this scaffolding protein.

Conclusions

While the NMR structure demonstrates that the CASKIN2 SH3 domain is folded, its cleft has suffered two substitutions that prevent it from binding typical polyproline ligands. This observation led us to additionally survey and describe other SH3 domains in the Protein Data Bank that may have similarly lost their ability to promote protein-protein interactions.

     

Prediction of binding sites of peptide recognition domains: an application on Grb2 and SAP SH2 domains

Determination of the binding motif and identification of interaction partners of the modular domains such as SH2 domains can enhance our understanding of the regulatory mechanism of protein-protein interactions. We propose here a new computational method to achieve this goal by integrating the orthogonal information obtained from binding free energy estimation and peptide sequence analysis. We performed a proof-of-concept study on the SH2 domains of SAP and Grb2 proteins. The method involves the following steps: (1) estimating the binding free energy of a set of randomly selected peptides along with a sample of known binders; (2) clustering all these peptides using sequence and energy characteristics; (3) extracting a sequence motif, which is represented by a hidden Markov model (HMM), from the cluster of peptides containing the sample of known binders; and (4) scanning the human proteome to identify binding sites of the domain. The binding motifs of the SAP and Grb2 SH2 domains derived by the method agree well with those determined through experimental studies. Using the derived binding motifs, we have predicted new possible interaction partners for the Grb2 and SAP SH2 domains as well as possible interaction sites for interaction partners already known. We also suggested novel roles for the proteins by reviewing their top interaction candidates.     

Multiple domains of Ruk/CIN85/SETA/CD2BP3 are involved in interaction with p85alpha regulatory subunit of PI 3-kinase

Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins.     

The 1.1 A resolution crystal structure of the p130cas SH3 domain and ramifications for ligand selectivity

The Crk-associated tyrosine kinase substrate p130cas (CAS) is a docking protein containing an SH3 domain near its N terminus, followed by a short proline-rich segment, a large central substrate domain composed of 15 repeats of the four amino acid sequence YxxP, a serine-rich region and a carboxy-terminal domain, which possesses consensus binding sites for the SH2 and SH3 domains of Src (YDYV and RPLPSPP, respectively). The SH3 domain of CAS mediates its interaction with several proteins involved in signaling pathways such as focal adhesion kinase (FAK), tyrosine phosphatases PTP1B and PTP-PEST, and the guanine nucleotide exchange factor C3G. As a homolog of the corresponding Src docking domain, the CAS SH3 domain binds to proline-rich sequences (PxxP) of its interacting partners that can adopt a polyproline type II helix. We have determined a high-resolution X-ray structure of the recombinant human CAS SH3 domain. The domain, residues 1-69, crystallized in two related space groups, P2(1) and C222(1), that provided diffraction data to 1.1 A and 2.1 A, respectively. The crystal structure shows, in addition to the conserved SH3 domain architecture, the way in which the CAS characteristic amino acids form an atypically charged ligand-binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by the CAS SH3 domain. The structure enables modelling of the docking interactions to its ligands, for example from focal adhesion kinase, and supports structure-based drug design of inhibitors of the CAS-FAK interaction.     

Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from 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 which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120^cbl). Like other SH3 domains, BTK SH3 domain consists of five -strands packed in two -sheets forming a -barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218–271 and the p120^cbl peptide residues 6–12 of the complex was 0.87 Å (±0.16 Å) for the backbone heavy atoms (N, C, and C) and 1.64 Å (±0.16 Å) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between 4 and 5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120^cbl binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of 4, 5 and the helix-like loop) exhibits weaker affinity for the p120^cbl peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA.     

Implications of SH3 domain structure and dynamics for protein regulation and drug design

SH3 Domains provide interesting targets for investigations of protein structure and dynamics because of their compact size and importance for signal transduction. The present review summarizes recent research investigating SH3 domain structure and dynamics, the discovery of novel SH3 domains, the role of SH3 domains in disease, and progress in targeting SH3 domains for the development of novel therapeutics. Particular emphasis is placed on the unfolding/refolding characteristics of SH3 domains and the potential importance of these processes for regulation of signal transduction.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline‐rich domain‐containing proteins such as Sos1. To define the difference in Grb2‐associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST‐Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST‐Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST‐Grb2 SH2 proteins exclusively bound to the p46^Shc, p52^Shc, and p66^Shc are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N‐SH3 and C‐SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2‐mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. J. Cell. Biochem. 84: 150–155, 2002. © 2001 Wiley‐Liss, Inc.     

The interaction between the adaptor protein APS and Enigma is involved in actin organisation

APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.     

Two phosphorylation-independent sites on the p85 SH2 domains bind A-Raf kinase.

Src homology 2 (SH2) domains mediate phosphotyrosine (pY)-dependent protein:protein interactions involved in signal transduction pathways. We have found that the SH2 domains of the 85-kDa alpha subunit (p85) of phosphatidylinositol 3-kinase (PI3 kinase) bind directly to the serine/threonine kinase A-Raf. In this report we show that the p85 SH2:A-Raf interaction is phosphorylation-independent. The affinity of the p85 C-SH2 domain for A-Raf and phosphopeptide pY751 was similar, raising the possibility that a p85:A-Raf complex may play a role in the coordinated regulation of the PI3 kinase and Raf-MAP kinase pathways. We further show that the p85 C-SH2 domain contains two distinct binding sites for A-Raf; one overlapping the phosphotyrosine-dependent binding site and the other a separate phosphorylation-independent site. This is the first evidence for a second binding site on an SH2 domain, distinct from the phosphotyrosine-binding pocket.     

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.     

Induction of cytochrome c release and apoptosis by Hck-SH3 domain-mediated signalling requires caspase-3

The function of key components of signal transduction, the Src family tyrosine kinases is dependent on catalytic activity as well as on intermolecular interaction achieved by their SH2 and SH3 modular domains. We have analyzed the effect of overexpression of the hematopoietic cell kinase (Hck) and its N-terminal unique and SH3 domains on cell survival. Overexpression of the N-terminal unique and SH3 domains (Hck-USH3) induced about 25% of expressing Cos-1 cells to undergo apoptosis 30 hrs after transfection. The full length p59 and p56 forms and the unique domain alone induced low levels of cell death. The unique and SH3 domain of a closely related kinase, Lyn did not induce apoptosis. Overexpression of a mutant USH3 domain (Gly Ala), that disrupts membrane localization, did not induce high level of apoptosis. Cells overexpressing Hck-USH3 showed activation of caspase-3 and release of cytochrome c from mitochondria into cytosol. Caspase-3 defective MCF-7 cells were resistant to apoptosis and cytochrome c release induced by Hck-USH3, which were restored by introducing the caspase-3 gene. These results suggest that Hck SH3 domain mediated signalling at the plasma membrane triggers a pathway leading to caspase-3 dependent cyto- chrome c release and apoptosis.