Zhang Yajie, Oh Hyunju, Burton Robert A, Burgner John W, Geahlen Robert L, Post Carol Beth
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11760-5. doi: 10.1073/pnas.0708583105. Epub 2008 Aug 8.
The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2(pm)) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2(pm). Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2(pm) are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2(pm) to dp-ITAM (1.8 microM) is significantly higher than that for the interaction between dp-ITAM and 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 change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed.
脾酪氨酸激酶（Syk）蛋白酪氨酸激酶在通过B细胞抗原受体（BCR）进行的信号传导中起主要作用。Syk通过其串联的SH2结构域与BCR复合物的双磷酸化免疫受体酪氨酸基激活基序（dp-ITAM）相互作用，从而与受体结合。位于两个远离磷酸酪氨酸结合位点的SH2结构域之间的Tyr-130发生磷酸化后，Syk会从受体上解离。为了理解这种解离的结构基础，我们研究了野生型串联SH2区域（tSH2）和变体串联SH2区域（tSH2(pm)）的结构和动力学特征，其中tSH2(pm)中的Tyr-130被Glu取代，从而在该位置永久引入负电荷。核磁共振异核弛豫实验、剩余偶极耦合测量和分析超速离心揭示了tSH2和tSH2(pm)在流体动力学行为上的显著差异。尽管tSH2中的两个SH2结构域紧密结合，但tSH2(pm)中的两个结构域部分解偶联，并且在溶液中以更快的相关时间翻转。此外，tSH2(pm)与dp-ITAM结合的平衡解离常数（1.8 microM）显著高于dp-ITAM与tSH2之间相互作用的解离常数，但接近于单酪氨酸磷酸化肽与单个SH2结构域结合的解离常数。实验数据和流体动力学计算均表明，在残基130上引入负电荷后，结构域间的接触丧失且相对取向发生变化。我们提出了一种远程结构机制，通过该机制Y130的磷酸化负调节Syk与免疫受体的相互作用。
