Grb2 SH3 binding to peptides from Sos: evaluation of a general model for SH3-ligand interactions

Abstract

Background: Grb2 acts as an adaptor protein in the transduction of signals from receptor tyrosine kinases to Ras. It binds to phosphotyrosine on the cytoplasmic tail of cell-surface receptors via its central SH2 domain, and to its immediate downstream target, Sos, via two SH3 domains. The basis of the Grb2-Sos interaction is not fully understood. We previously proposed a model for SH3 domain binding specificity, based on two solution structures of the Src SH3 domain complexed with high-affinity ligands, in which the ligands are bound in a polyproline type II conformation in two distinct orientations, class I and class II. Here, we have used this model to predict the identity and orientation of Grb2 SH3 ligands in the human Sos protein.

Results: Six contiguous fragments from the carboxy-terminal portion of hSos (amino acids 1000-1333), each containing a single potential SH3 binding site, were expressed in E. coli as GST fusion proteins. Four of these proteins were predicted to associate with SH3 domains. The amino-terminal Grb2 SH3 domain was shown to bind strongly to only these four fragments.

Conclusions: We have used a general model for SH3-ligand interactions to predict the nature of Grb2 SH3 interactions with the hSos protein. Comparison of the four hSos sequences that bind Grb2 revealed a preference for the PXXPXR motif, consistent with the predicted class II-type binding interaction. The interaction between Grb2 and hSos peptides is predominantly via the amino-terminal SH3 domain, although the carboxy-terminal SH3 domain may increase the overall stability of the Grb2-hSos complex.