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SRC homology 2 domain proteins binding specificity: from combinatorial chemistry to cell-permeable inhibitors

Wavreille, Anne-Sophie Marie
http://rave.ohiolink.edu/etdc/view?acc_num=osu1164738844

Abstract Details

2006, Doctor of Philosophy, Ohio State University, Chemistry.
Protein-protein interactions form the molecular basis of a wide variety of cellular processes. A large fraction of these interactions are mediated by small modular domains, which bind to short peptide motifs in their partner proteins. However, for the vast majority of these modular domains, their binding specificity and interacting partners remain unknown. This work presents a chemical/bioinformatic approach to the identification of the binding proteins of the Src Homology 2 domain (SH2). First, a combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY+6 positions for the four SH2 domains of protein tyrosine phosphatases SHP-1 and SHP-2. The screening results were confirmed by surface plasmon resonance analysis, stimulation assays and in vitro pull-down experiments. This study reveals that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues at these positions. Similar residues at the pY+4 to pY+6 positions are also preferred by SHP-1 and SHP-2 C-SH2 domains, although their influence on the overall binding affinities is much smaller compared with the N-SH2 domains. Second, our chemical/bioinformatic approach was applied to identify the binding proteins of tensin. Another pY peptide library was screened against the tensin SH2 domain to determine the peptide motifs that bind to this domain. The peptide motifs were employed to search protein databases for potential tensin-binding proteins, which were subsequently confirmed (or disproved) by in vitro pull-down and coimmunoprecipitation assays. This procedure identified phosphoinositide-dependent kinase-1 (PDK-1) and downstream of tyrosine kinase 2 (Dok-2) as novel tensin-binding proteins. In addition, a cell-permeable pY peptide was designed as tensin SH2 domain inhibitor, which caused the disruption of actin filaments in NIH 3T3 cells. Third, the sequence specificity of other SH2 domains (c-Src, Grb2, Syk, ZAP-70 and SLAP) were established. Finally, our strategy was applied to other modular domains: chromodomains, another type of library: a cyclic peptide library and a different screening: a live cell binding assay.
Dehua Pei (Advisor)
215 p.
Chemistry, Biochemistry
SH2 domain; Binding specificity; SHP-1; SHP-2; Tensin; peptide library; protein interaction; cell-permeable inhibitor

Recommended Citations

Citations

  • Wavreille, A.-S. M. (2006). SRC homology 2 domain proteins binding specificity: from combinatorial chemistry to cell-permeable inhibitors [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1164738844

    APA Style (7th edition)

  • Wavreille, Anne-Sophie. SRC homology 2 domain proteins binding specificity: from combinatorial chemistry to cell-permeable inhibitors. 2006. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1164738844.

    MLA Style (8th edition)

  • Wavreille, Anne-Sophie. "SRC homology 2 domain proteins binding specificity: from combinatorial chemistry to cell-permeable inhibitors." Doctoral dissertation, Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1164738844

    Chicago Manual of Style (17th edition)

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© 2006, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.