Mechanism of G protein Activation by Ric-8A

Ric-8A激活G蛋白的机制

• 批准号: 9751877
• 负责人: Stephen R Sprang
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751877
• 关键词: Active Sites; Address; Adenylate Cyclase; Adjuvant; Adopted; Armadillo Repeat; Binding; Biogenesis; Biological Assay; Cell division; Cell membrane; Cells; Cellular Metabolic Process; Chemicals; Collaborations; Complex; Coupled; Cryoelectron Microscopy; Crystallization; Cytoplasm; Data; Disease; Electric Conductivity; Electrons; Embryo; Embryonic Development; Enzymes; Event; Funding; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genetic Transcription; Goals; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Heteronuclear NMR; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Human Development; Image; Intercept; Ion Channel; Knock-out; Knowledge; Laboratories; Life; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Medicine; Microscopic; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monte Carlo Method; Movement; Mutagenesis; Nucleotides; Phosphorylation; Physiological Processes; Process; Property; Protein Isoforms; Proteins; Reaction; Research; Resolution; Roentgen Rays; Role; Route; Sampling; Second Messenger Systems; Sequence Homology; Signal Transduction; Structure; Testing; Transducers; Ubiquitin; Ursidae Family; Vesicle; Work; X-Ray Crystallography; beta pleated sheet; casein kinase II; cell motility; experimental study; extracellular; molecular dynamics; particle; prevent; protein activation; protein folding; receptor; response; scaffold; small molecule; trafficking

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.

项目摘要R01GM105993 R01-GM105993的这种续订的目的是确定该结构和动态特性 RIC-8a与异三聚体G蛋白GI的α亚基之间的复合物（GαI1）。 蛋白质调节细胞代谢，分泌，电导率，基因转录，细胞分裂和 细胞运动性，因此对于真核生活至关重要。 G蛋白的不调节与 癌症和一系列与普通医学相关的疾病。而大多数过程由 异三聚合物G蛋白发生在细胞膜上，最近的研究表明，Gα亚基（Gα）也 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 对于胚胎开发至关重要。在此过程中，RIC-8a是Gα的关键调节剂。 RIC-8A是鸟嘌呤 核苷酸交换因子（GEF）通过催化鸟苷二磷酸的交换来激活Gα （GDP）三磷酸三磷酸（GTP）的GDP。该反应中的中间体是 无核苷酸Gα：RIC-8A复合物。描述RIC-8A与 Gα·GDP是理解RIC-8A如何激活Gα的关键。 RIC-8A也是促进适当的伴侣 细胞中Gα的折叠。该提案的两个目标将检验以下假设：GαI1和可能的RIC-8A 在复合物GαI1中采样多种构象状态：RIC-8A，并利用结构信息到 询问GEF和伴侣活动在机械上是不可分割的，还是是不同的功能 可以分离。 RIC-8A受蛋白激酶CK2调节的机制也将是 调查。 AIM 1是通过X射线晶体学确定RIC-8a及其与GαI1的复合物的结构， 前者在磷酸化和非磷酸化状态下。骆驼单链重链 可变域将用作结晶调节器。协作异核NMR实验将 以独立的方法来定义RIC-8A和GαI之间的界面并确定 构象状态之间过渡的残留物。将进行GEF和伴侣活动的测定 评估从结构研究中假设残留物的作用，作为一个或两个的介体 这些活动。 AIM 2将使用小角度X射线散射确定RIC-8A：GαI1的全局结构 和冷冻电子显微镜。使用分子动力学对较低分辨率结构进行建模 RIC-8A和GαI1的X射线结构的蒙特卡洛模拟将用于检测和识别多个 复合体的构象状态。