ORIENTATION OF PEPTIDE-FRAGMENTS FROM SOS PROTEINS BOUND TO THE N-TERMINAL SH3 DOMAIN OF GRB2 DETERMINED BY NMR-SPECTROSCOPY

NMR spectroscopy has been used to characterize the protein-protein int eractions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complex ed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to resid ues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular inte ractions between the peptide and C-13-N-15-labeled SH3 domain were ide ntified in half-reverse-filtered 2D and 3D NOESY experiments. Assignme nts for the protons involved in interactions between the peptide and t he SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuter ated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 doma in is defined by the side chains of specific aromatic residues (Tyr(7) , Phe(9), TrP(36), Tyr(52)) that form two hydrophobic subsites contact ing the side chains of the peptide Leu(4) and Leu(7) residues. An adja cent negatively charged subsite on the SH3 surface is likely to intera ct with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 doma in structure in the complex is well-defined (backbone RMSD of 0.56+/-0 .21 calculated over the backbone N, C alpha, and C atoms of residues 1 -54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation. Relative to the SH3 domain bindi ng site, the polypeptide backbone of the peptide in the complex is pos itioned in the opposite orientation compared to that recently describe d for the synthetic peptide (RKLPPRP)-PI3K SH3 domain complex [Yu et a l. (1994) Cell 76, 933-945]. The mSos peptide-mGrb2 N-terminal SH3 com plex has a dissociation constant of 54 mu M as determined by isotherma l titration calorimetry. Another mSos peptide (VPPPVPPRRR) exhibits ti ghter binding (3.5 mu M dissociation constant) with the N-terminal Grb 2 SH3 domain and appears to bind in a similar manner on the basis of N OESY experiments.