title: Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy.
abstract: NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact 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 domain 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
authors.forename: M
authors.initials: M
authors.lastname: Wittekind
authors.forename: C
authors.initials: C
authors.lastname: Mapelli
authors.forename: B T
authors.initials: BT
authors.lastname: Farmer
authors.forename: K L
authors.initials: KL
authors.lastname: Suen
authors.forename: V
authors.initials: V
authors.lastname: Goldfarb
authors.forename: J
authors.initials: J
authors.lastname: Tsao
authors.forename: T
authors.initials: T
authors.lastname: Lavoie
authors.forename: M
authors.initials: M
authors.lastname: Barbacid
authors.forename: C A
authors.initials: CA
authors.lastname: Meyers
authors.forename: L
authors.initials: L
authors.lastname: Mueller
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003196:Computer Graphics; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D009842:Oligopeptides; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; C090505:Sos-A protein, mouse