Recognition of lysine-rich peptide ligands by murine cortactin SH3 domain: CD, ITC, and NMR studies.
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.
Rubini
Chiara
C
Institute of Biomolecular Chemistry of CNR, Padova, Italy.
Ruzza
Paolo
P
Spaller
Mark R
MR
Siligardi
Giuliano
G
Hussain
Rohanah
R
Udugamasooriya
D Gomika
DG
Bellanda
Massimo
M
Mammi
Stefano
S
Borgogno
Andrea
A
Calderan
Andrea
A
Cesaro
Luca
L
Brunati
Anna M
AM
Donella-Deana
Arianna
A
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
D051356:Cortactin; D008024:Ligands; D010455:Peptides; D008239:Lysine