Characterization of structural and dynamic properties of Syk tandem SH2 domain and its interaction with diphosphorylated-ITAM important in B cell signaling regulation
Abstract
The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds to the receptor via its tandem pair of SH2 domains (tSH2) interacting with a doubly-phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) on components of the BCR complex. Upon phosphorylation of Tyr130, which lies between the two SH2 domains, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we substituted Tyr130 with Glu (Y130E) to permanently introduce a negative charge at this position. We then used various biophysical methodologies to study the structural and dynamic characteristics of the wild type and variant tSH2, and the data were combined to define a mechanism by which the phosphorylation of Y130 regulates the interaction of Syk with immune receptors. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of wild type tSH2 (Sykwt tSH2) and Y130E tSH2. While Syk tSH2 undergoes conformational exchange at free state, the two SH2 domains are still motionally coupled. Those in the Y130E tSH2, on the other hand, tumble separately in solution. In addition, the equilibrium dissociation constant for the binding of Y130E tSH2 to dp-ITAM (1.8 μM) is significantly higher than that measured for the interaction between dp-ITAM and Sykwt tSH2, but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and coordinated motion upon the introduction of a negative charge on residue 130. The interaction between Syk protein with dual-phosphorylated ITAM peptide is, therefore, mainly controlled via the alteration of inter-domain flexibility of tSH2 domain, which complements the previous biological data showing the negative regulatory role of Y130 phosphorylation on Syk signaling pathway.
Degree
Ph.D.
Advisors
Post, Purdue University.
Subject Area
Biochemistry|Biophysics
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