Molecular and cellular analysis of Grb2 SH3 domain mutants: Interaction with Sos and dynamin

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measur ed between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform bind ing proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions . Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt f olded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overal l stability of these domains by inducing an equilibrium between a folded an d an unfolded form. The complex formed between the peptide VPPPVPPRRR, deri ved from Sos, and the E40T mutant was shown to have the same 3D structure a s that described for the wild-type SH3 domain. However, the VPPPVPPRRR pept ide adopts a slightly different orientation when it is complexed with the Y 7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mut ants were tested on a cellular homogenate by means of a far-Western blot an alysis. In these conditions, the P49L mutant was shown to be devoid of affi nity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decreas e of affinity for both Sos and dynamin, while the E40T mutant exhibited a d ecrease of affinity only for dynamin. These results support the existence o f two binding sites between dynamin and the Grb2 N-SH3 domain. (C) 1999 Aca demic Press.