Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.