BIOPHYSICAL STUDIES OF SRC HOMOLOGY 2 DOMAINS

本研究的目的是研究Src同源2（SH 2）结构域在两个蛋白酪氨酸激酶（PTK）系统：Syk和Src中结合的结构、热力学和动力学性质。SH 2结构域是在特定序列环境中识别并结合磷酸化酪氨酸残基的蛋白质结构域，从而允许蛋白质募集到信号蛋白的酪氨酸磷酸化的结构域上。磷酸酪氨酸的C-末端序列对于SH 2结构域特异性识别磷酸肽靶标是必不可少的。Syk和Src家族PTK在免疫细胞中协作以介导B-和T-细胞受体的信号传导：Src家族激酶，其仅含有单个SH 2结构域，通过磷酸化酪氨酸残基的串联重复序列响应受体活化，所述串联重复序列又充当Syk激酶的两个SH 2结构域的SH 2结构域对接位点。我们的工作目标有三个方面：1）使用Src SH 2结构域作为模型建立SH 2结构域中肽结合特异性的能量原理，2）使用Syk的串联-SH 2结构域作为模型检查当存在多于一个SH 2结构域时SH 2结构域之间的协同相互作用，以及3）从长远来看，研究SH 2结构域在全长激酶中的功能。本提案中计划的研究旨在通过SH 2结构域剖析酪氨酰磷酸酪氨酸大分子识别的决定因素，使用一系列方法，包括调查溶液（离子，pH值）和温度的作用，肽和蛋白质的定点诱变，以及X射线晶体学。

The aim of this proposal is to study the structural, thermodynamic, and kinetic properties of binding of Src Homology 2 (SH2) domains in two protein tyrosine kinase (PTK) systems: Syk and Src. SH2 domains are protein domains which recognize and bind phosphorylated tyrosine residues in specific sequence contexts, thereby allowing protein recruitment onto tyrosine-phosphorylated sties of signaling proteins. Sequences C-terminal to the phosphotyrosine are essential for specific recognition of phosphopeptide targets by SH2 domains. Syk and Src family PTKs cooperate in immune cells to mediate signaling by B- and T-cell receptors: Src family kinases which contain only a single SH2 domain response to receptor activation by phosphorylating tandem repeats of tyrosine residues, which in turn serve as SH2 domain-docking sites for the two SH2 domains of the Syk kinase. The goals of our work are three-fold: 1) establish the energetic principles of peptide binding specificity in SH2 domains using the Src SH2 domain as a model, 2) examine the cooperative interactions between SH2 domains when more than one SH2 domain is present using the tandem- SH2 domain of Syk as a model, and 3) in the longer term, examine the function of SH2 domains in the context of the full-length kinase. The study planned in this proposal aims at dissecting the determinants of macromolecular recognition at tyrosyl-phosphotyrosine by SH2 domains, using an array of methodologies that include the investigation of the role of solutions (ions, pH) and temperature, site-directed mutagenesis of both peptides and proteins, and x-ray crystallography.