STRUCTURAL BASIS OF G PROTEIN MEDIATED SIGNALING

G 蛋白介导的信号传导的结构基础

• 批准号: 6386736
• 负责人: David G Lambright
• 金额: $ 25.14万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386736
• 关键词: G protein; X ray crystallography; conformation; crystallization; enzyme structure; enzyme substrate complex; guanine nucleotide exchange factors; guanosinetriphosphatases; intermolecular interaction; physical model; precipitation; protein binding; protein sequence; protein structure function; site directed mutagenesis; structural biology; surface plasmon resonance; thermodynamics

GTP-binding proteins (G proteins) mediate critical steps in a variety of signaling and regulatory pathways. The function of G proteins in diverse biological processes depends on their ability to cycle between inactive (GDP-bound) and active (GTP-bound) conformations in a precisely regulated manner. Aberrant forms of G proteins have been implicated in the pathogenesis of variety of disease states including cancer. Consequently, G proteins and the factors that regulate their activity are potential targets for pharmacological interdiction. The experiments described in this proposal address the mechanism by which Mss4, a 13 kDa exchange factor selective for a subset of Rab family GTPases, regulates the activation of the monomeric G protein Rab3a which functions in neuronal signaling. The experimental approach will combine high resolution structural studies by X-ray crystallography with biochemical and mutational experiments in order to identify with the principal stereochemical determinants of Rab3a activation and regulation by Mss4 as well as the inactive (GDP-bound) and active (GMPPNP-bound) forms of Rab3a, the respective crystal structures will be solved and refined at high resolution. These studies will reveal the conformational changes associated with activation and provide a structural basis for the function of Mss4 and Rab3a in neuronal signaling. In parallel, mutations in Rab3a and Mss4 will be introduced to probe the binding interaction and determine the basis for the selectivity in the interaction between Mss4 and Rab family GTPases. The results, when interpreted in the context of the Mss4 and Raba3a crystal structures, will provide a clear picture of the structural and functional determinants that govern specificity and lead to the release of nucleotide. Finally, screens will be conducted for co-crystals of Mss4 bound to Rab3a or other Rav family proteins. Comparison with structural and biochemical data for other systems including EF-Tu/EF-Ts will distinguish features unique to the Mss4/Rab3a system from those that may be global determinants for nucleotide exchange. In addition, the structure of the GMPPNP-bound form of Rab3a will provide a basis for exploring the regulatory interaction between the active form of Rav proteins and specific effector proteins in the fusion/docking complex which mediates vesicle fusion with target membranes in exocytosis.

GTP结合蛋白（G蛋白）介导多种生物学过程中的关键步骤。 信号传导和调节途径。 G蛋白的功能 不同的生物过程取决于它们在不同的生物体之间循环的能力， 非活性（GDP结合）和活性（GTP结合）构象在一个精确的 规范的方式。 G蛋白的异常形式与 包括癌症在内的多种疾病的发病机制。 因此，G蛋白和调节其活性的因子 是药物阻断的潜在目标。 实验 本提案中描述的方法解决了Mss 4，一个13 kDa 对Rab家族GTP酶的子集具有选择性的交换因子，调节 单体G蛋白Rab 3a的激活， 神经信号 该实验方法将联合收割机高度 用X射线晶体学和生物化学方法进行解析结构研究 和突变实验来认同 Rab 3a激活和Mss 4调节的立体化学决定簇 以及非活性（GDP结合）和活性（GMPPNP结合）形式的 Rab 3a中，将求解并细化相应的晶体结构， 高分辨率。 这些研究将揭示构象变化 与激活相关，并为 Mss 4和Rab 3a在神经元信号传导中的功能。与此同时， Rab 3a和Mss 4中的突变将被引入以探测结合 相互作用，并确定选择性的基础， Mss 4和Rab家族GTP酶之间的相互作用。 结果，当 在Mss 4和Raba 3a晶体结构的上下文中解释， 将提供一个清晰的结构和功能的图片 决定因素，支配特异性，并导致释放 核苷酸 最后，筛选Mss 4的共晶 结合Rab 3a或其他Rav家族蛋白。 与结构比较 包括EF-Tu/EF-Ts在内的其他系统的生化数据将 将Mss 4/Rab 3a系统特有的特征与可能 是核苷酸交换的全局决定因素。 此外该 Rab 3a的GMPPNP结合形式的结构将为 探索活性形式的Rav 融合/对接复合物中的蛋白质和特异性效应蛋白 其在胞吐作用中介导囊泡与靶膜的融合。