NUCLEAR-MAGNETIC-RESONANCE SOLUTION STRUCTURE OF THE GROWTH-FACTOR RECEPTOR-BOUND PROTEIN-2 SRC HOMOLOGY-2 DOMAIN

A family of NMR solution structures of the growth factor receptor-boun d protein 2 (Grb2) SH2 domain has been determined by heteronuclear mul tidimensional NMR, Proton, nitrogen, and carbon chemical shift assignm ents have been made for the SH2 domain of Grb2. Assignments were made from a combination of homonuclear two-dimensional and N-15- and C-13-e dited three-dimensional spectra at pH 6.2 and 298 K. Structure-induced proton and carbon secondary shifts were calculated and used to facili tate the spectral assignment process. NOE, scalar coupling, secondary chemical shift. and amide proton exchange data were used to characteri ze the secondary structural elements and hydrogen-bonding network in t he Grb2 SH2 domain. The three-dimensional structure of the Grb2 SH2 do main was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data. The rmsd for the 24 calculat ed structures to the mean structure of the Grb2 SH2 domain was 0.75 An gstrom for backbone and 1.28 Angstrom for all heavy atoms. The three-d imensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eigh t beta-strands, six strands that make up two contiguous antiparallel b eta-sheets, and two strands that form two short parallel beta-sheets. The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains. The hydrophobic binding pocke t of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket. Struc tural implications far the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.