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.

Molecular aspects of multivalent engagement between Syk and FcεRIγ

“…4). In particular, there is a decrease in the number of contacts between the N-SH2 and C-SH2 domains in the presence of the mutation, which is consistent with the experimentally-observed partial decoupling of both SH2 domains in the mutant (Zhang et al, 2008;Feng and Post, 2016;Roy et al, 2016).…”

“…MD simulations provided insufficient information on the influence of the Y130E substitution on helical stability, due to limited sampling even at microsecond time scales. Motivated by experimental observations that Y130E does disrupt helical stability (Zhang et al, 2008;Feng and Post, 2016;Roy et al, 2016), we computed inter-SH2 distance distribution from REMD simulations of the interdomain A linker. Only the linker was included in these simulations in order to enhance the sampling of adopted conformations and to evaluate the impact of the Y130E mutation.…”

“…We specially focus on Syk recruitment after phosphorylation at site Y130 in interdomain A (Keshvara et al, 1998), since Y130E substitution in Syk resulted in a phosphomimetic recombinant protein with reduced binding of its tandem SH2 domains to phosphorylated ITAMs in co-immunoprecipitation studies (Keshvara et al, 1997;Zhang et al, 2008). This was initially attributed to the structural destabilization of interdomain A and the resulting partial decoupling of both SH2 domains (Zhang et al, 2008;Feng and Post, 2016;Roy et al, 2016). However, high resolution imaging studies in Syk-deficient cells reconstituted with Syk Y130E recombinant protein revealed two interesting observations: 1) Syk recruitment to FcεRI aggregates is retained even for Y130E phosphomimetic, and 2) impaired downstream signaling correlates with an increased off-rate for FcεRI-Syk-Y130E interactions (Schwartz et al, 2017).…”

Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics

“…The importance of the longer-lived interactions in signal propagation is shown by introduction of a Y130E mutation within the I-A domain of Syk. Phosphorylation of Y130 is proposed as a form of negative-feedback regulation, because it has been shown to destabilize binding of Syk tandem SH2 domains to phosphorylated ITAMs (pITAMs) ( Zhang et al , 2008 ; Feng and Post, 2016 ). We find that Syk-Y130E is still recruited to FcεRI aggregates, but its interactions are more transient ( k s = 0.87 s −1 ) and markedly less efficient at transphosphorylation.…”

Differential Mast Cell Outcomes Are Sensitive to FcεRI-Syk Binding Kinetics

“…The importance of the longer-lived interactions in signal propagation is shown by introduction of a Y130E mutation within the I-A domain of Syk. Phosphorylation of Y130 is proposed as a form of negative feedback regulation since it has been shown to destabilize binding of Syk tandem SH2 domains to phosphorylated ITAMs (pITAM) (Feng and Post, 2015;Zhang et al, 2008). We find that Syk-Y130E is still recruited to FcRI aggregates, but its interactions are more transient (ks = 0.87 s -1 ) and markedly less efficient at transphosphorylation.…”