Solution structure of the SH2 domain of Grb2/Ash complexed with EGF receptor-derived phosphotyrosine-containing peptide.
1H, 13C, and 15N NMR resonances of the SH2 domain of Grb2/Ash in both the free form and the form complexed with a phosphotyrosine-containing peptide derived from the EGF receptor were assigned by analysis of multi-dimensional, double- and triple-resonance NMR experiments. From the chemical shift changes of individual residues upon peptide binding, the binding site for the 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 the EGFR peptide does not bind to Grb2/Ash SH2 in an extended conformation. This was supported by analysis of the binding affinity of mutants where residues on the BG and EF loops were changed to alanine. The present results are consistent with the recently reported structures of Grb2/Ash SH2 complexed with BCR-Abl and Shc-derived phosphotyrosine containing peptides, where the peptide forms a turn conformation. This shows that the specific conformation of the phosphotyrosine-containing sequence is required for the SH2 binding responsible for downstream signaling.
Tsuchiya
S
S
Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, 113-8613, Japan.
Ogura
K
K
Hatanaka
H
H
Nagata
K
K
Terasawa
H
H
Mandiyan
V
V
Schlessinger
J
J
Aimoto
S
S
Ohta
H
H
Inagaki
F
F
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics; D051380:GRB2 Adaptor Protein; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D019000:Phosphotyrosine / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012996:Solutions; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D046911:Macromolecular Substances; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D019000:Phosphotyrosine; D066246:ErbB Receptors