Mechanism of G protein Activation by Ric-8A

项目摘要R01GM105993 R01-GM105993更新的目标是确定结构和动态性能 Ric-8A与异三聚体G蛋白Gi的α亚基(Gαi1)之间的复合体。杂三聚体G 蛋白质调节细胞新陈代谢、分泌、电导、基因转录、细胞分裂和 细胞的运动性，因此对真核生物来说是必不可少的。G蛋白的错误调节与 癌症和一系列与普通医学相关的其他疾病。虽然大多数进程都由 异源三聚体G蛋白存在于细胞膜上，最近的研究表明Gα亚基(Gα)还 控制细胞质中的某些事件。其中重要的是不对称的细胞分裂，这是 对胚胎发育至关重要。在这一过程中，RIC-8A是G-α的关键调节因子。RIC-8A是一种鸟嘌呤 通过催化鸟苷二磷酸交换来激活Gα的核苷酸交换因子 G-α活性部位的三磷酸鸟苷的GDP值。这个反应中的中间体是 无核苷酸Gα：RIC-8A复合体。描述当RIC-8A绑定到时发生的结构变化 Gα·gdp是理解RIC-8A如何激活Gα的关键。RIC-8A也是一种伴侣，促进适当的 Gα在细胞内的折叠。这项提议的两个目标将检验Gαi1和可能的RIC-8A的假设 对络合物Gαi1：RIC-8A中的多个构象状态进行采样，并利用结构信息 问全球环境基金和陪护活动在机制上是不可分割的，还是不同的职能 可以脱钩。Ric-8A受蛋白激酶CK2调节的机制也将是 调查过了。目的1是用X射线结晶学方法确定Ric-8A及其与G-αI1的配合物的结构， 前者既处于磷酸化状态又处于非磷酸化状态。驼状单链重链 可变结构域将被用作结晶助剂。合作的异核核磁共振实验将 作为独立方法进行，以定义RIC-8A和GαI之间的接口并确定 构象状态间转换中的残基。将对全球环境基金和陪护活动进行分析 为了评估结构研究中假设的残基作为一种或两种物质的中介的作用 这些活动。Aim 2将利用小角X射线散射确定Ric-8A：GαI1的全局结构 和低温电子显微镜。利用分子动力学和分子动力学对低分辨结构进行模拟 Ric-8A和Gαi1的X射线结构的蒙特卡罗模拟将用于检测和识别多个 络合物的构象状态。

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.