Publication

Article Metrics

Citations


Online attention

Recognition of lysine-rich peptideligands by murine cortactin SH3 domain: CD, ITC and NMR studies

DOI: 10.1002/bip.21350 DOI Help

Authors: Chiara Rubini (Institute of Biomolecular Chemistry of CNR, Padova Unit, 35131 Padova, Italy) , Paolo Ruzza (Institute of Biomolecular Chemistry of CNR) , Mark R. Spaller (Department of Chemistry, Wayne State University) , D. Gomika Udugamasooriya (Department of Chemistry, Wayne State University) , Massimo Ballanda (Department of Chemical Sciences, University of Padova) , Steafno Mammi (Institute of Biomolecular Chemistry of CNR) , Andrea Borgogno (Institute of Biomolecular Chemistry of CNR) , Andrea Calderan (Institute of Biomolecular Chemistry of CNR) , Luca Cesaro (Institute of Biomolecular Chemistry of CNR) , Anna M. Brunati (Department of Biological Chemistry, University of Padova) , Arianna Donella-deana (Department of Biological Chemistry, University of Padova) , Rohanah Hussain (Diamond Light Source) , Giuliano Siligardi (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biopolymers

State: Published (Approved)
Published: November 2009

Abstract: 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 394403) and S1 (Shank2 1168-1189) peptides. GST-SH3(m-cort) exhibits the highest affinity toward peptide Si, 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. (C) 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 298-306, 2010.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: B23-Circular Dichroism