SOLUTION STRUCTURE OF THE HUMAN PP60(C-SRC) SH2 DOMAIN COMPLEXED WITH A PHOSPHORYLATED TYROSINE PENTAPEPTIDE

Human pp60(c-src) is a cellular nonreceptor tyrosine kinase that participates in cytosolic signal transduction and has been implicated in the development of malignant tumors in the human breast and colon. Signal transduction is mediated by highly specific interactions between the SH2 domain and receptor phosphorylated tyrosine binding motifs. To elucidate the molecular conformation and interactions in solution, a family of highly resolved nuclear magnetic resonance (NMR) structures was determined for the src SH2 domain complexed with a high-affinity phosphorylated pentapeptide, acetyl-pYEEIE-OH. The 23 structures, generated with a distance geometry (DG) and a dynamical simulated annealing (SA) procedure, satisfied 2072 experimental restraints derived from a variety of multifrequency/multidimensional and isotope-filtered NMR data. Superimposition of residues 143-245 upon the mean coordinate set yielded an atomic rmsd of 0.58 +/- 0.09 Angstrom for the N, C alpha, C' atoms and 1.04 +/- 0.08 for all the nonhydrogen atoms. Residues in the ordered secondary structure regions superimpose to 0.29 +/- 0.04 Angstrom for the N, C alpha, C' and 0.73 +/- 0.08 Angstrom for all the non-hydrogen atoms. The angular order parameter calculated for the cp, II, angles was > 0.9 for 81 of the 106 protein residues. The main protein conformational features are three antiparallel P-strands that traverse a compact core with an cl-helix on each side of the core near the N- and C-termini. The observed intermolecular nuclear Overhauser effects (NOE) from the pY, +1E, and +3I residues positioned the ligand in an extended conformation across the SH2 domain surface with the pY and +3I side chains inserted into the protein binding pockets. In general, the protein conformation is consistent with previously reported structures of different SH2 domain complexes determined by X-ray crystallography. However, inter- or intramolecular interactions involving the guanidinium side chains of the solvated R alpha A2 or the buried R beta B5 were not observed at pH = 5.5 or 7.0. If such interactions exist in solution, the absence of any confirming data probably arises from rapid exchange with solvent and/or undetermined dynamic components. Thus, the unrestrained R alpha A2 side chain did not show an amino-aromatic interaction or a hydrogen bond to the -1 carbonyl oxygen as observed in the crystal structures. This result is consistent with the solution structure of a different SH2 domain complex. A more detailed comparison between the crystal structure and the NMR-derived solution structures of the same src SH2 domain complex is presented. Small conformational differences for ligand residues exposed to observed crystal contacts and at the +3I binding region are discussed.