MEASUREMENT OF THE BINDING OF TYROSYL PHOSPHOPEPTIDES TO SH2 DOMAINS - A REAPPRAISAL

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathw ays. Attempts to understand the determinants of specificity and select ivity of these interactions have prompted many binding studies that ha ve used several techniques. Some discrepancies, in both the absolute a nd relative values of the dissociation constants for particular intera ctions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed th ree previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry, We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dis sociation constant (K-d) of 600 nM; a phosphopeptide based on one bind ing site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subu nit with a K-d of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a K-d of 4 mu M. In additi on, we demonstrate that avidity effects that result from the dimerizat ion of glutathione S-transferase fusion proteins with SH2 domains coul d be responsible for overestimates of affinities for these interaction s previously studied by surface plasmon resonance.