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Tony Pawson - One of the best experts on this subject based on the ideXlab platform.
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structural basis for specific binding of the gads SH3 Domain to an rxxk motif containing slp 76 peptide a novel mode of peptide recognition
Molecular Cell, 2003Co-Authors: Donna M Berry, Tony Pawson, Piers Nash, Jane C Mcglade, Shawn S C LiAbstract:Abstract The SH3 Domain, which normally recognizes proline-rich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 Domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 docking protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 Domain and upon binding adopts a unique structure characterized by a right-handed 3 10 helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 Domains.
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A potential SH3 Domain-binding site in the Crk SH2 Domain
Journal of Biological Chemistry, 1996Co-Authors: Mordechai Anafi, Michael K. Rosen, Gerald D. Gish, Tony PawsonAbstract:Abstract The Src homology 2 (SH2) Domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding. Using the yeast two-hybrid system, this region of the Crk-II SH2 Domain was found to interact with a subset of SH3 Domains, notably the Abl SH3 Domain. Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140c-abl tyrosine kinase. In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase-Abl SH3 Domain fusion protein was very weak. However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 Domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 Domain. NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 Domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction. These results suggest that the proline-rich insert in the Crk SH2 Domain constitutes an SH3 Domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 Domain.
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the v src SH3 Domain binds phosphatidylinositol 3 kinase
Molecular and Cellular Biology, 1993Co-Authors: L E M Marengere, C A Koch, Tony PawsonAbstract:Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 Domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 Domain, but not the SH2 Domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 Domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 Domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 Domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.
Shawn S C Li - One of the best experts on this subject based on the ideXlab platform.
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the SH3 Domain a family of versatile peptide and protein recognition module
Frontiers in Bioscience, 2008Co-Authors: Tomonori Kaneko, Lei Li, Shawn S C LiAbstract:Abstract Src homology 3 (SH3) Domains were initially characterized as a prevalent protein module that recognizes proline-rich sequences, in particular those containing a PxxP motif. Recent studies have shown that the specificity and cellular function of SH3 Domains are far more diverse than previously appreciated. Despite lacking distinguishing features, the ligand-binding surface of an SH3 Domain can be molded to accommodate a variety of peptide ligands. Moreover, certain SH3 Domains are capable of using surfaces distinct from the canonical ligand-binding site to engage a peptide or protein. The identification of novel motifs and Domains recognized by the SH3 Domain greatly expands the ligand pool and cellular function for this family. However, this also imposes the question as to how the specificity of the hundreds of human SH3 Domains is regulated in a cell to ensure their proper functions. Here we review literature on the specificity of SH3 Domains, with an emphasis on the structural basis of ligand recognition, and discuss mechanisms employed by SH3 Domain-containing proteins to execute defined cellular functions through highly regulated SH3-ligand interactions.
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structural basis for specific binding of the gads SH3 Domain to an rxxk motif containing slp 76 peptide a novel mode of peptide recognition
Molecular Cell, 2003Co-Authors: Donna M Berry, Tony Pawson, Piers Nash, Jane C Mcglade, Shawn S C LiAbstract:Abstract The SH3 Domain, which normally recognizes proline-rich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 Domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 docking protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 Domain and upon binding adopts a unique structure characterized by a right-handed 3 10 helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 Domains.
David Baker - One of the best experts on this subject based on the ideXlab platform.
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Structural and kinetic characterization of the simplified SH3 Domain FP1.
Protein Science, 2003Co-Authors: Qian Yi, Ponni Rajagopal, Rachel E. Klevit, David BakerAbstract:The simplified SH3 Domain sequence, FP1, obtained in phage display selection experiments has an amino acid composition that is 95% Ile, Lys, Glu, Ala, Gly. Here we use NMR to investigate the tertiary structure of FP1. We find that the overall topology of FP1 resembles that of the src SH3 Domain, the hydrogen-deuterium exchange and chemical shift perturbation profiles are similar to those of naturally occurring SH3 Domains, and the 15N relaxation rates are in the range of naturally occurring small proteins. Guided by the structure, we further simplify the FP1 sequence and compare the effects on folding kinetics of point mutations in FP1 and the wild-type src SH3 Domain. The results suggest that the folding transition state of FP1 is similar to but somewhat less polarized than that of the wild-type src SH3 Domain.
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Prediction and structural characterization of an independently folding substructure in the src SH3 Domain.
Journal of Molecular Biology, 1998Co-Authors: Qian Yi, Ponni Rajagopal, Rachel E. Klevit, Christopher Bystroff, David BakerAbstract:Previous studies of the conformations of peptides spanning the length of the a-spectrin SH3 Domain suggested that SH3 Domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 Domain predicted to adopt a nativelike structure, a type II b-turn bridging unpaired b-strands, that was not contained intact in any of the SH3 Domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3 JNHa coupling constant patterns were indicative of a type II b-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding. # 1998 Academic Press
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important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 Domain
Nature Structural & Molecular Biology, 1998Co-Authors: Viara P Grantcharova, David S Riddle, Jed V Santiago, David BakerAbstract:Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two β-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 Domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 Domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.
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folding dynamics of the src SH3 Domain
Biochemistry, 1997Co-Authors: Viara P Grantcharova, David BakerAbstract:The thermodynamics and kinetics of folding of the chicken src SH3 Domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 Domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4−3.7 M). Estimates of the free energy of unfold...
Ismail Moarefi - One of the best experts on this subject based on the ideXlab platform.
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structural basis for SH3 Domain mediated high affinity binding between mona gads and slp 76
The EMBO Journal, 2003Co-Authors: Maria Harkiolaki, Marc Lewitzky, Robert J C Gilbert, Eyvonne Jones, Roland P Bourette, Guy Mouchiroud, Holger Sondermann, Ismail Moarefi, Stephan M FellerAbstract:SH3 Domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 Domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 Domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 Domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 Å. The peptide lacks the canonical SH3 Domain binding motif P–x–x–P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C β-barrel. The central R–x–x–K motif of the peptide forms a 310 helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 Domain functions.
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activation of the src family tyrosine kinase hck by SH3 Domain displacement
Nature, 1997Co-Authors: Ismail Moarefi, John Kuriyan, M Lafevrebernt, Frank Sicheri, Morgan Huse, W T MillerAbstract:The protein Hck is a member of the Src family of non-receptor tyrosine kinases which is preferentially expressed in haematopoietic cells of the myeloid and B-lymphoid lineages1,2. Src kinases are inhibited by tyrosine-phosphorylation at a carboxy-terminal site3–9. The SH2 Domains of these enzymes play an essential role in this regulation by binding to the tyrosine-phosphorylated tail8–11. The crystal structure of the downregulated form of Hck has been determined12 and reveals that the SH2 Domain regulates enzymatic activity indirectly; intramolecular interactions between the SH3 and catalytic Domains appear to stabilize an inactive form of the kinase. Here we compare the roles of the SH2 and SH3 Domains in modulating the activity of Hck in an investigation of the C-terminally phosphorylated form of the enzyme. We show that addition of the HIV-1 Nef protein, which is a high-affinity ligand for the Hck SH3 Domain, to either the downregulated or activated form of Hck causes a large increase in Hck catalytic activity. The intact SH3-binding motif in Nef is crucial for Hck activation. Our results indicate that binding of the Hck SH3 Domain by Nef causes a more marked activation of the enzyme than does binding of the SH2 Domain, suggesting a new mechanism for regulation of the activity of tyrosine kinases.
Michael Jaye - One of the best experts on this subject based on the ideXlab platform.
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Identification of a Src SH3 Domain binding motif by screening a random phage display library
Journal of Biological Chemistry, 1994Co-Authors: Christopher Cheadle, Victoria South, Richard Howk, George A. Ricca, Victoria J. South, Stephen French, Yuri Ivashchenko, George H Searfoss, Michael JayeAbstract:A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) Domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 Domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding Domain core motif displayed strong binding to both the Src and to the Fyn SH3 Domains, but failed to bind to the SH3 Domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 Domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 Domains and those containing SH3 binding motifs.
