SOLUTION STRUCTURE OF THE GRB2 N-TERMINAL SH3 DOMAIN COMPLEXED WITH A10-RESIDUE PEPTIDE DERIVED FROM SOS - DIRECT REFINEMENT AGAINST NOES,J-COUPLINGS AND H-1 AND C-13 CHEMICAL-SHIFTS

Refined ensembles of solution structures have been calculated for the N-terminal SH3 domain of Grb2 (N-SH3) complexed with the ac-VPPPVPPRRR -nh2 peptide derived from residues 1135 to 1144 of the mouse SOS-1 seq uence. NMR spectra obtained from different combinations of both C-13-N -15-labeled and unlabeled N-SHS and SOS peptide fragment were used to obtain stereo-assignments for pro-chiral groups of the peptide, angle restraints via heteronuclear coupling constants, and complete H-1, C-1 3, and N-15 resonance assignments for both molecules. One ensemble of structures was calculated using conventional methods while a second en semble was generated by including additional direct refinements agains t both H-1 and C-13(alpha)/C-13(beta) chemical shifts. In both ensembl es, the protein:peptide interface is highly resolved, reflecting the i nclusion of 110 inter-molecular nuclear Overhauser enhancement (NOE) d istance restraints. The first and second peptide-binding sub-sites of N-SH3 interact with structurally well-defined portions of the peptide. These interactions include hydrogen bonds and extensive hydrophobic c ontacts. In the third highly acidic sub-site, the conformation of the peptide Arg8 side-chain is partially ordered by a set of NOE restraint s to the Trp36 ring protons. Overall, several Lines of evidence point to dynamical averaging of peptide and N-SH3 side-chain conformations i n the third subsite. These conformations are characterized by transien t charge stabilized hydrogen bond interactions between the peptide arg inine side-chain hydrogen bond donors and either single, or possibly m ultiple, acceptor(s) in the third peptide-binding sub-site. (C) 1997 A cademic Press Limited.