Structure, kinetics, and backbone dynamics of the SH2 domain of human proto-oncogene protein Vav1 bound to a doubly phosphorylated Syk peptide
Abstract
Syk-dependent activation of NFAT signaling in B cells depends on phosphorylation of tyrosine residues in Syk linker B. Phosphorylation of both Y324 and Y346 of this motif creates the site for recognition of the C-terminal Src homology 2 domain of PLC-γ (PLCC SH2) and Vav1 SH2. The NMR structure of the PLCC SH2 complexed with a doubly phosphorylated linker B peptide, pYpY, revealed a novel mode of SH2 recognition through a large loop rearrangement to accommodate the atypical second phosphotyrosine in the non-canonical phosphotyrosine binding pocket. The newly determined NMR structure of the Vav1 SH2 domain in complex with the identical peptide is compared to the structures of the PLCC SH2-pYpY complex and Vav1 SH2 bound to a singly phosphorylated peptide derived from SLP-76. Unlike PLCC SH2, Vav1 SH2 undergoes only small conformational changes relative to the unbound state. The recognition role of a lysine residue at the βD3 position conserved in all SH2 domains that interact with the Syk linker B region containing dual phosphotyrosines is further confirmed by the structure of the Vav1 SH2-pYpY complex. NMR 15N relaxation data imply the existence of conformational exchange in the free state, from which it is suggested that conformational heterogeneity play a role in the Vav1 SH2-phosphopeptide binding. The exchange processes were not observed in the CPMG-based relaxation dispersion experiments, indicating that the motions in the absence of peptide are on the time scale faster than millisecond. The interactions between singly phosphorylated Syk peptides and Vav1 SH2 were also investigated. Syk linker B peptides with single phosphorylation at either Y342 (pYY) or Y346 (YpY) result in twofold and tenfold reduction in binding affinity for pYpY, respectively. Biochemical studies show that wild-type Syk, Y346F, and Y342F variants induce different levels of Vav1 phosphorylation and NFAT activation. Strong linear relationships between binding affinities and signal transduction responses explain how Syk phosphorylation regulates the downstream signaling. The residues with large chemical shift perturbations are in agreement with those in direct contact with the peptide and/or undergoing conformational changes upon binding pYpY. Chemical shift perturbations also suggest that the pYpY-bound and pYY-bound complexes of Vav1 SH2 are more similar to each other than the YpY-bound complex. The PLCC SH2 and Vav1 SH2 domains recognize the same doubly phosphorylated Syk linker B sequence via distinct binding modes. Phosphorylation of Y342 and Y346 and the differences in the recognition and mechanism of the two competing proteins could be functionally important in the proper regulation of B cell signaling.
Degree
Ph.D.
Advisors
Post, Purdue University.
Subject Area
Biochemistry|Biophysics
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