The phosphorylation of a linker region between two tandem SH2 domains of Syk tyrosine kinase regulates the binding affinity for Syk association with membrane receptors. 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, to a substantially lower-affinity binding of only one SH2 domain. We examined the postulated switch to a single-SH2-domain binding mode using NMR to monitor site-specific binding to each SH2 domain and using Syk variants engineered to have IA regions that differ in conformational flexibility. With the residue-level power of NMR, we refuted the premise that phosphorylation restricts binding to only one SH2 domain, and measured the energetics of inter-molecular binding at each SH2 site along with an intra-molecular binding or isomerization step. A physical basis for understanding the effects of linker phosphorylation on domain structure and the isomerization step of binding is lacking. Simulations are being used to explore the multiple domain behavior of the tandem SH2s and basis for allosteric regulation of binding by phosphorylation.

Multiple SH2 domains: Binding and dynamics

251st National Meeting (2016)

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