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 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)

[1]  L. Kay,et al.  A Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlations in H2O Samples of Proteins , 1993 .

[2]  N. Jullian,et al.  Solution structure of GAP SH3 domain by 1H NMR and spatial arrangement of essential Ras signaling‐involved sequence. , 1994, The EMBO journal.

[3]  S. Schreiber,et al.  Solution structure of the SH3 domain of Src and identification of its ligand-binding site. , 1992, Science.

[4]  M. Wigler,et al.  Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. , 1993, Science.

[5]  Nanxin Li,et al.  Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling , 1993, Nature.

[6]  D. Bowtell,et al.  Identification of murine homologues of the Drosophila son of sevenless gene: potential activators of ras. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[7]  F. Richards,et al.  Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition , 1994, Nature Structural Biology.

[8]  R. Weinberg,et al.  The pathway to signal achievement , 1993, Nature.

[9]  Michael J. Eck,et al.  Structure of the regulatory domains of the Src-family tyrosine kinase Lck , 1994, Nature.

[10]  S. Schreiber,et al.  Biased combinatorial libraries: novel ligands for the SH3 domain of phosphatidylinositol 3-kinase , 1993 .

[11]  V. Sasisekharan.  Structure of poly-l-proline. II , 1959 .

[12]  T Pawson,et al.  SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. , 1991, Science.

[13]  S. Schreiber,et al.  1H and 15N assignments and secondary structure of the PI3K SH3 domain , 1993, FEBS letters.

[14]  Hongtao Yu,et al.  Structural basis for the binding of proline-rich peptides to SH3 domains , 1994, Cell.

[15]  J. Schlessinger,et al.  Solution structure of the SH3 domain of phospholipase C-γ , 1993, Cell.

[16]  Andrea Musacchio,et al.  Crystal structure of a Src-homology 3 (SH3) domain , 1992, Nature.

[17]  M. Friedrichs,et al.  An automated procedure for the assignment of protein 1HN, 15N, 13Cα, 1Hα, 13Cβ and 1Hβ resonances , 1994 .

[18]  T. Pawson,et al.  Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors , 1993, Molecular and cellular biology.

[19]  Stuart L. Schreiber,et al.  Structure of the Pl3K SH3 domain and analysis of the SH3 family , 1993, Cell.

[20]  Paul A. Keifer,et al.  Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity , 1992 .

[21]  M. Saraste,et al.  Crystal structure of the SH3 domain in human Fyn; comparison of the three‐dimensional structures of SH3 domains in tyrosine kinases and spectrin. , 1993, The EMBO journal.

[22]  K. Constantine,et al.  Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy , 1993, Journal of biomolecular NMR.

[23]  R. Weinberg,et al.  Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation , 1993, Nature.

[24]  M. Sternberg,et al.  Left-handed polyproline II helices commonly occur in globular proteins. , 1993, Journal of molecular biology.

[25]  Andrea Musacchio,et al.  High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides , 1994, Nature Structural Biology.

[26]  U. Banerjee,et al.  Genetic dissection of a neurodevelopmental pathway: Son of sevenless functions downstream of the sevenless and EGF receptor tyrosine kinases , 1991, Cell.

[27]  I. Campbell,et al.  Solution structure and ligand-binding site of the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase , 1993, Cell.

[28]  L. Mueller,et al.  Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins , 1994, Journal of biomolecular NMR.

[29]  T. Pawson,et al.  The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1 , 1993, Nature.

[30]  A. Ullrich,et al.  The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling , 1992, Cell.