Solution structure of the Grb2 N-terminal SH3 domain complexed with a ten-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 sequence. 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-13, and N-15 resonance assignments for both molecules. One ensemble of structures was calculated using conventional methods while a second ensemble was generated by including additional direct refinements against both H-1 and C-13(alpha)/C-13(beta) chemical shifts. In both ensembles, the protein:peptide interface is highly resolved, reflecting the inclusion of 110 inter-molecular nuclear Overhauser enhancement (NOE) distance 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 contacts. In the third highly acidic sub-site, the conformation of the peptide Arg8 side-chain is partially ordered by a set of NOE restraints to the Trp36 ring protons. Overall, several Lines of evidence point to dynamical averaging of peptide and N-SH3 side-chain conformations in the third subsite. These conformations are characterized by transient charge stabilized hydrogen bond interactions between the peptide arginine side-chain hydrogen bond donors and either single, or possibly multiple, acceptor(s) in the third peptide-binding sub-site. (C) 1997 Academic Press Limited.