Directed discovery of bivalent peptide ligands to an SH3 domain |
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Authors: | Ferguson Monique R Fan Xiuzhen Mukherjee Munia Luo Jinquan Khan Raza Ferreon Josephine C Hilser Vincent J Shope Robert E Fox Robert O |
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Institution: | Department of Human Biological Chemistry and Genetics, and Sealy Center for Structural Biology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA. |
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Abstract: | The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains. |
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Keywords: | SH3 domain signal transduction phage display combinatorial library peptide ligand bivalent ligand NMR spectroscopy non-PXXP binding site |
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