ALTERNATIVE MODES OF TYROSYL PHOSPHOPEPTIDE BINDING TO A SRC FAMILY SH2 DOMAIN - IMPLICATIONS FOR REGULATION OF TYROSINE KINASE-ACTIVITY

Src homology 2 (SH2) domains interact with proteins containing phospho rylated tyrosine residues and as such play a key role in mediating tyr osine kinase signal transduction, Determination of how these interacti ons maintain specificity is central to understanding the mechanism of this intracellular signal processing. In the Src family tyrosine kinas es specificity is enhanced by a form of regulation based on binding of a phosphotyrosine, pY, and its proximal amino acid sequence from the C-terminus to the SH2 domain of the same protein (autoregulation) or t o a similar protein (homodimeric regulation). Activation of the protei n is accomplished by removal of this regulatory interaction by competi tion from a ''specific'' interacting ligand. We adopt the SH2 domain f rom a member of the Src family, Fyn (whose predominant physiological r ole is in initiation of signals from the T-cell receptor complex), to explore the differences in structural, thermodynamic, and kinetic dete rminants of regulatory and specific interactions using tyrosyl phospho peptides based on the C-terminus and on a putative physiological inter acting species from the hamster middle-sized tumor antigen. The specif ic peptide interacts with micromolar affinity via embedding the pY and an isoleucine residue (in the pY + 3 position) in two deep pockets. T his leads to a large favorable enthalpic contribution to free energy. The regulatory peptide interacts in the pY pocket which forms a pivot for the rest of the molecule which is dynamic. These structural data f or the regulatory peptide are supported by the observation of a more f avorable entropic term and a complex mode of binding revealed by kinet ic analysis.