Crystal structure of the pleckstrin homology-phosphotyrosine binding (PH-PTB) targeting region of insulin receptor substrate 1

We have determined the crystal structure at 2.3-Angstrom resolution of an a mino-terminal segment of human insulin receptor substrate 1 that encompasse s its pleckstrin homolog (PH) and phosphotyrosine binding (PTB) domains. Bo th domains adopt the canonical selen-stranded beta-sandwich PR domain fold. The domains are closely associated, with a 720-Angstrom(2) contact surface buried between them that appears to he stabilized by ionic, hydrophobic, a nd hydrogen bonding interactions. The nonconserved 46-residue linker betwee n the domains is disordered. The PTB domain peptide binding site is fully e xposed on the molecular surface, as is a large cationic patch at the base o f the PH domain that is a likely binding site for the head groups of phosph atidylinositol phosphates, Binding assays confirm that phosphatidylinositol phosphates bind the PH domain, but not the PTB domain. Ligand binding to t he PH domain does not alter PTB domain interactions, and vice versa. The st ructural and accompanying functional data illustrate how the two binding do mains might act cooperatively to effectively increase local insulin recepto r substrate 1 concentration at the membrane and transiently fix the recepto r and substrate, to allow multiple phosphorylation reactions to occur durin g each union.