STRUCTURAL BASIS OF G PROTEIN MEDIATED SIGNALING

GTP结合蛋白(G蛋白)在多种生物中起关键作用 信号和调控通路。G蛋白在细胞周期调控中的作用 不同的生物过程取决于它们之间循环的能力 非活性构象(GDP结合)和活性构象(GTP结合) 规范的方式。G蛋白的异常形式被认为与 包括癌症在内的多种疾病状态的发病机制。 因此，G蛋白和调节其活性的因素 是药物阻断的潜在靶点。这些实验 本提案中描述的机制解决了Mss 4，一个13 kDa 对Rab家族GTP酶的子集选择性的交换因子，调节 发挥作用的单体G蛋白Rab3a的激活 神经元信号。实验方法将结合高 X-射线结晶学结合生物化学的分辨结构研究 和突变实验，以便认同校长 Rab3a激活的立体化学决定因素及Mss-4的调节 以及非活动(GDP绑定)和活动(GMPPNP绑定)形式 Rab3a，各自的晶体结构将在 高分辨率。这些研究将揭示构象变化。 与激活相关联，并为 Mss4和Rab3a在神经元信号转导中的作用。同时， 将引入Rab3a和Mss4的突变来探测结合 交互作用，并确定在 Mss-4与Rab家族GTP酶的相互作用。结果，当 在Mss 4和Raba3a晶体结构的上下文中解释， 将提供结构和功能的清晰图景 控制特异性并导致释放的决定因素 核苷酸。最后，将对Mss 4的共晶进行筛选 与Rab3a或其他RAV家族蛋白结合。与结构的比较 以及包括EF-Tu/EF-TS在内的其他系统的生化数据将 区分Mss 4/Rab3a系统独有的功能与可能 是核苷酸交换的全球决定因素。此外， GMPPNP结合形式的Rab3a的结构将为 探讨RAV活性形式之间的调控相互作用 融合/对接复合体中的蛋白质和特异性效应蛋白 它在胞吐作用中介导囊泡与靶膜融合。

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.