Mechanism of G protein Activation by Ric-8A

项目总结R 01 GM 105993 R 01-GM 105993更新的目的是确定 Ric-8A与异源三聚体G蛋白Gi（Gαi1）α亚基之间的复合物。异三聚体G 蛋白质调节细胞代谢、分泌、电导率、基因转录、细胞分裂和细胞增殖。 细胞运动，因此对真核生物至关重要。G蛋白的失调与 癌症和一系列与普通医学相关的其他疾病。虽然大多数过程由 异源三聚体G蛋白存在于细胞膜上，最近的研究表明G α亚基（Gα）也 控制细胞质中的某些事件。其中重要的是不对称细胞分裂， 对胚胎发育至关重要。Ric-8A是Gα在此过程中的关键调节因子。Ric-8A是鸟嘌呤 一种通过催化鸟苷二磷酸交换来激活Gα的核苷酸交换因子（GEF (GDP)在Gα的活性位点上的三磷酸鸟苷（GTP）。这个反应的中间体是 无核苷酸Gα：Ric-8A复合物。描述了当Ric-8A结合到 Gα·GDP是理解Ric-8A如何激活Gα的关键。Ric-8A也是一种伴侣， 细胞内Gα折叠。该提议的两个目的将检验Gαi1和可能的Ric-8A 对复合物Gαi1：Ric-8A中的多种构象状态进行采样，并利用结构信息 问全球环境基金和监护人活动在机制上是否不可分割，还是属于不同的职能， 可以解耦。Ric-8A受蛋白激酶CK 2调节的机制也将被详细讨论。 研究了目的1：用X射线晶体学方法测定Ric-8A及其与Gαi1配合物的结构， 前者处于磷酸化和非磷酸化状态。骆驼科动物单链重链 可变结构域将用作结晶助剂。协同异相NMR实验将 作为一种独立的方法进行，以确定Ric-8A和GαI之间的界面，并确定 构象状态之间的过渡残基。将对全环基金和监护人活动进行分析 评估从结构研究中假设的残基作为以下一种或两种介质的作用： 这些活动。目标2将使用小角X射线散射确定Ric-8A：Gαi1的整体结构 和冷冻电子显微镜。使用分子动力学和分子动力学模拟低分辨率结构 利用Ric-8A和Gαi1的X射线结构进行蒙特卡罗模拟， 复合物的构象状态。

PROJECT SUMMARY R01GM105993 The goal of this renewal of R01-GM105993 is to determine the structure and dynamic properties of the complex between Ric-8A and the alpha subunit of the heterotrimeric G protein Gi (Gαi1). Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to eukaryotic life. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (Gα) also control certain events in the cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of Gα in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that activates Gα by catalyzing the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of Gα. The intermediate in this reaction is the nucleotide-free Gα:Ric-8A complex. Describing the structural changes that occur when Ric-8A binds to Gα·GDP is key to understanding how Ric-8A activates Gα. Ric-8A is also a chaperone that promotes proper folding of Gα in cells. The two aims of this proposal will test the hypothesis that Gαi1 and possibly Ric-8A sample multiple conformational states in the complex Gαi1:Ric-8A and to make use of structural information to ask whether the GEF and chaperone activities are mechanistically inseparable, or are distinct functions that can be decoupled. The mechanism by which Ric-8A is regulated by protein kinase CK2 will also be investigated. Aim 1 is to determine the structure of Ric-8A and its complex with Gαi1 by X-ray crystallography, with the former in both phosphorylated and non-phosphorylated states. Camelid single chain heavy chain variable domains will be used as crystallization adjuvants. Collaborative heteronuclear NMR experiments will be conducted as an independent approach to define the interface between Ric-8A and GαI and to identify residues in transition among conformational states. Assays of GEF and chaperone activities will be conducted to assess the role of residues that are hypothesized from structural studies as mediators of one or both of these activities. Aim 2 will determine the global structure of Ric-8A:Gαi1 using small angle X-ray scattering and cryo-electron microscopy. Modeling of the lower resolution structures using molecular dynamics and Monte Carlo simulations with X-ray structures of Ric-8A and Gαi1 will be used to detect and identify multiple conformational states of the complex.