PH TITRATION STUDIES OF AN SH2 DOMAIN-PHOSPHOPEPTIDE COMPLEX - UNUSUAL HISTIDINE AND PHOSPHATE PK(A) VALUES

Electrostatic interactions in a complex of the phospholipase C-gamma(1 ) C-terminal SH2 domain with a high-affinity binding phosphopeptide re presenting the sequence around Tyr 1021 of the beta platelet-derived g rowth factor receptor were studied by pK(a) determination of various t itratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His beta D4) and the phosphoty rosine (pTyr) phosphate group show pK(a) values significantly lower th an average for these residue types in proteins. The reduced pK(a) of t hese two groups is due to the proximity of the highly positively charg ed pTyr binding pocket. The unusual pK(a) of His beta D4 is also due t o burial from solvent in a hydrogen-bonding network that appears neces sary for the positioning of arginine residues involved in pTyr binding . Mutation of the analogous histidine in other SH2 domains has been sh own to abrogate pTyr binding. In addition to these large shifts in pK( a) values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second he lix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domai n-phosphopeptide complex confirms previous descriptions of the roles o f different arginines in the structure and function of this protein.