title: Recognition of lysine-rich peptide ligands by murine cortactin SH3 domain: CD, ITC, and NMR studies.
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 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.
authors.affiliation: Institute of Biomolecular Chemistry of CNR, Padova, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Rubini
authors.forename: Paolo
authors.initials: P
authors.lastname: Ruzza
authors.forename: Mark R
authors.initials: MR
authors.lastname: Spaller
authors.forename: Giuliano
authors.initials: G
authors.lastname: Siligardi
authors.forename: Rohanah
authors.initials: R
authors.lastname: Hussain
authors.forename: D Gomika
authors.initials: DG
authors.lastname: Udugamasooriya
authors.forename: Massimo
authors.initials: M
authors.lastname: Bellanda
authors.forename: Stefano
authors.initials: S
authors.lastname: Mammi
authors.forename: Andrea
authors.initials: A
authors.lastname: Borgogno
authors.forename: Andrea
authors.initials: A
authors.lastname: Calderan
authors.forename: Luca
authors.initials: L
authors.lastname: Cesaro
authors.forename: Anna M
authors.initials: AM
authors.lastname: Brunati
authors.forename: Arianna
authors.initials: A
authors.lastname: Donella-Deana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002942:Circular Dichroism; D051356:Cortactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D018909:src Homology Domains
chemical_list: D051356:Cortactin; D008024:Ligands; D010455:Peptides; D008239:Lysine