Chen Chih-Hong, Piraner Dan, Gorenstein Nina M, Geahlen Robert L, Beth Post Carol
Department of Medicinal Chemistry and Molecular Pharmacology, and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, 47907.
Biopolymers. 2013 Nov;99(11):897-907. doi: 10.1002/bip.22371.
The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling.
脾酪氨酸激酶（Syk）是B细胞信号传导中的一种核心酪氨酸激酶，它与Vav SH2结构域的结合受Syk连接子B中两个紧密相邻的酪氨酸Y342和Y346磷酸化的控制。先前的研究表明，单磷酸化和双磷酸化形式在信号传导中均发挥作用。双磷酸化形式的结构确定了对磷酸酪氨酸的一种新识别方式，即两个磷酸酪氨酸同时与Vav SH2结构域结合，一个在典型的pTyr口袋中，另一个在中央β折叠另一侧的特异性口袋中。尚不清楚特异性口袋是否能独立于与pTyr口袋结合的磷酸酪氨酸而结合磷酸酪氨酸。为了解决这一知识空白，我们确定了Vav1 SH2与模拟Syk单磷酸化 - Y346形式且Y342未磷酸化的肽段（SykLB - YpY）之间复合物的结构。核磁共振（NMR）数据确凿地表明，磷酸酪氨酸的识别在两个口袋之间发生了交换；磷酸化的pY346结合Vav1 SH2的特异性口袋，未磷酸化的Y342占据通常的pTyr结合口袋。结合所有三种单磷酸化和双磷酸化肽段形式时，化学位移发生了几乎相同的变化；然而，来自高内部流动性区域残基的SykLB - YpY的化学位移扰动略小，这表明内部运动与结合亲和力相关。这种包括磷酸酪氨酸与Vav SH2特异性口袋交换结合的差异识别增加了SH2结构域在调节细胞信号传导中蛋白质 - 蛋白质相互作用时可能的磷酸酪氨酸结合方式。
