Solution structure of the SH2 domain of Grb2/Ash complexed with EGF receptor-derived phosphotyrosine-containing peptide

H-1, C-13, and N-15 NMR resonances of the SH2 domain of Grb2/Ash in both th e free form and the form complexed with a phosphotyrosine-containing peptid e derived from the EGF receptor were assigned by analysis of multi-dimensio nal, double- and triple-resonance NMR experiments. From the chemical shift changes of individual residues upon peptide binding, the binding site for t he peptide was mapped on the structure of Grb2/Ash SH2, The peptide was not recognized by the groove formed by the BG and EF loops, suggesting that th e EGFR peptide does not bind to Grb2/Ash SH2 in an extended conformation. T his was supported by analysis of the binding affinity of mutants where resi dues on the BG and EF loops were changed to alanine, The present results ar e consistent with the recently reported structures of Grb2/Ash SH2 complexe d with BCR-Abl and She-derived phosphotyrosine containing peptides, where t he peptide forms a turn conformation. This shows that the specific conforma tion of the phosphotyrosine-containing sequence is required for the SH2 bin ding responsible for downstream signaling.