BIOPHYSICAL STUDIES OF SRC HOMOLOGY 2 DOMAINS

SRC 同源性 2 域的生物物理学研究

• 批准号: 6351313
• 负责人: GABRIEL WAKSMAN
• 金额: $ 20.32万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351313
• 关键词: X ray crystallography; acidity /alkalinity; biological signal transduction; biophysics; ionic strengths; phosphatidylinositol 3 kinase; phosphorylation; protein binding; protein sequence; protein tyrosine kinase; site directed mutagenesis; temperature; thermodynamics

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.

本提案的目的是研究两个蛋白酪氨酸激酶 (PTK) 系统：Syk 和 Src 中 Src 同源 2 (SH2) 结构域结合的结构、热力学和动力学特性。 SH2结构域是识别并结合特定序列背景中的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结构域上。磷酸酪氨酸 C 端序列对于 SH2 结构域特异性识别磷酸肽靶标至关重要。 Syk 和 Src 家族 PTK 在免疫细胞中合作，介导 B 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来响应受体激活，而酪氨酸残基又充当 Syk 激酶的两个 SH2 结构域的 SH2 结构域对接位点。我们工作的目标有三个：1) 使用 Src SH2 结构域作为模型，建立 SH2 结构域中肽结合特异性的能量原理；2) 使用 Syk 的串联 SH2 结构域作为模型，检查存在多个 SH2 结构域时 SH2 结构域之间的协同相互作用；3) 从长远来看，检查全长激酶背景下 SH2 结构域的功能。该提案中计划的研究旨在剖析 SH2 结构域对酪氨酰磷酸酪氨酸大分子识别的决定因素，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。