BIOPHYSICAL STUDIES OF SRC HOMOLOGY 2 DOMAINS

本研究的目的是研究两个蛋白酪氨酸激酶(PTK)系统中的Src同源2(SH2)结构域结合的结构、热力学和动力学性质。Sh2结构域是在特定的序列环境中识别和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酪氨酸磷酸化的信号蛋白上。磷酸酪氨酸的C末端序列对于SH2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在免疫细胞中相互协作，通过B细胞和T细胞受体介导信号传递：SRC家族激酶只包含一个SH2结构域，通过磷酸化酪氨酸残基的串联重复序列来响应受体的激活，而酪氨酸残基的串联重复序列又作为Syk激酶的两个SH2结构域的SH2结构域对接位置。我们的工作目标有三个：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Tandem-SH2结构域为模型，研究多个SH2结构域之间的协同相互作用；3)从长期来看，SH2结构域在全长激酶的背景下的功能。这项研究计划采用一系列方法，包括研究溶液(离子、pH)和温度的作用，多肽和蛋白质的定点突变，以及X射线结晶学，旨在剖析SH2结构域对酪氨酰磷酸酪氨酸大分子识别的决定因素。

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.