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Recognition of lysine‐rich peptide ligands by murine cortactin SH3 domain: CD, ITC, and NMR studies
Author(s) -
Rubini Chiara,
Ruzza Paolo,
Spaller Mark R.,
Siligardi Giuliano,
Hussain Rohanah,
Udugamasooriya D. Gomika,
Bellanda Massimo,
Mammi Stefano,
Borgogno Andrea,
Calderan Andrea,
Cesaro Luca,
Brunati Anna M.,
DonellaDeana Arianna
Publication year - 2009
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21350
Subject(s) - sh3 domain , isothermal titration calorimetry , cortactin , peptide , chemistry , circular dichroism , biochemistry , plasma protein binding , proto oncogene tyrosine protein kinase src , biophysics , microbiology and biotechnology , biology , signal transduction , cell , cytoskeleton
Cortactin is a ubiquitous actin‐binding protein that regulates various aspects of cell dynamics and is implicated in the pathogenesis of human neoplasia. The sequence of cortactin contains a number of signaling motifs and an SH3 domain at the C‐terminus, which mediates the interaction of the protein with several partners, including Shank2. A recombinant protein, comprising the murine cortactin SH3 domain fused to GST (GST‐SH3 m‐cort ), was prepared and used to assess the domain‐binding affinity of potential peptide‐ligands reproducing the proline‐rich regions of human HPK1 and Shank2 proteins. The key residues involved in the SH3 m‐cort domain recognition were identified by three different approaches: non‐immobilized ligand interaction assay by circular dichroism, isothermal titration calorimetry, and nuclear magnetic resonance. Our results show that the classical PxxPxK class II binding motif is not sufficient to mediate the interaction with GST‐SH3 m‐cort , an event that depends on the presence of additional basic residues located at either the N‐ or the C‐terminus of the PxxPxK motif. Especially effective in promoting the peptide binding is a Lys residue at the ‐5 position, a determinant present in both P2 (HPK1 394‐403) and S1 (Shank2 1168‐1189) peptides. GST‐SH3 m‐cort exhibits the highest affinity toward peptide S1, which contains additional Lys residues at the ‐3, ‐5, and ‐7 positions, indicating that the optimal consensus motif may be KPPxPxKxKxK. These results are supported by the in silico models of SH3 m‐cort complexed with P2 or S1, which highlight the domain residues that interact with the recognition determinants of the peptide‐ligand and cooperate in binding stabilization. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 298–306, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com