NMR structure of the N-SH2 of the p85 subunit of phosphoinositide 3-kinase complexed to a doubly phosphorylated peptide reveals a second phosphotyrosine binding site.
The N-terminal src homology 2 (SH2) domain of the p85 subunit of phosphoinositide 3-kinase (PI3K) has a higher affinity for a peptide with two phosphotyrosines than for the same peptide with only one. This unexpected result was not observed for the C-terminal SH2 from the same protein. NMR structural analysis has been used to understand the behavior of the N-SH2. The structure of the free SH2 domain has been compared to that of the SH2 complexed with a doubly phosphorylated peptide derived from polyomavirus middle T antigen (MT). The structure of the free SH2 domain shows some differences from previous NMR and X-ray structures. In the N-SH2 complexed with a doubly phosphorylated peptide, a second site for phosphotyrosine interaction has been identified. Further, line shapes of NMR signals showed that the SH2 protein-ligand complex is subject to temperature-dependent conformational mobility. Conformational mobility is also supported by the spectra of the ligand peptide. A binding model which accounts for these results is developed.
Weber
T
T
Institut für Biophysikalische Chemie, J. W. Goethe Universität, Frankfurt, Biozentrum N230, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
Schaffhausen
B
B
Liu
Y
Y
Günther
U L
UL
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D008024:Ligands; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012996:Solutions; D013696:Temperature; D013816:Thermodynamics; D018909:src Homology Domains
D000952:Antigens, Polyomavirus Transforming; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D012996:Solutions; C075879:polyoma peptide antigen MT162-176; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases