Insights into the allosteric regulation of Syk association with receptor ITAM, a multi-state equilibrium.

Abstract

The phosphorylation of interdomain A (IA), a linker region between tandem SH2 domains of Syk tyrosine kinase, regulates the binding affinity for association of Syk with doubly-phosphorylated ITAM regions of the B cell receptor. The mechanism of this allosteric regulation has been suggested to be a switch from the high-affinity bifunctional binding, mediated through both SH2 domains binding two phosphotyrosine residues of ITAM, to a substantially lower-affinity binding of only one SH2 domain. IA phosphorylation triggers the switch by inducing disorder in IA and weakening the SH2-SH2 interaction. The postulated switch to a single-SH2-domain binding mode is examined using NMR to monitor site-specific binding to each SH2 domain of Syk variants engineered to have IA regions that differ in conformational flexibility. The combined analysis of titration curves and NMR line-shapes provides sufficient information to determine the energetics of inter-molecular binding at each SH2 site along with an intra-molecular binding or isomerization step. A less favorable isomerization equilibrium associated with the changes in the SH2-SH2 conformational ensemble and IA flexibility accounts for the inhibition of Syk association with membrane ITAM regions when IA is phosphorylated, and refutes the proposed switch to single-SH2-domain binding. Syk localizes in the cell through its SH2 interactions, and this basis for allosteric regulation of ITAM association proposes for the first time a phosphorylation-dependent model to regulate Syk binding to alternate receptors and other signaling proteins that differ either in the number of residues separating ITAM phosphotyrosines or by having only one phosphotyrosine, a half ITAM.