BIOPHYSICAL STUDIES OF SRC HOMOLOGY 2 DOMAINS

本研究旨在研究Syk和Src两种蛋白酪氨酸激酶（PTK）系统中Src同源性2 （SH2）结构域结合的结构、热力学和动力学性质。SH2结构域是识别和结合特定序列背景下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酪氨酸磷酸化的信号蛋白上。磷酸酪氨酸的c端序列是SH2结构域特异性识别磷酸肽目标所必需的。Syk和Src家族PTKs在免疫细胞中合作介导B细胞和t细胞受体的信号传导：Src家族激酶仅包含一个SH2结构域，通过磷酸化酪氨酸残基的串联重复序列对受体激活做出反应，而酪氨酸残基反过来又作为Syk激酶两个SH2结构域的SH2结构域对接位点。我们的工作目标有三个方面：1)以Src SH2结构域为模型，建立SH2结构域中肽结合特异性的能量原理；2)以Syk的串联- SH2结构域为模型，研究多个SH2结构域存在时SH2结构域之间的合作相互作用；3)从长远来看，在全长激酶的背景下，研究SH2结构域的功能。本研究计划的目的是通过SH2结构域剖析酪氨酸-磷酸酪氨酸大分子识别的决定因素，使用一系列方法，包括研究溶液（离子、pH）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.