Mechanism of G protein Activation by Ric-8A

Ric-8A激活G蛋白的机制

基本信息

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
项目类别：
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

项目摘要

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 更新的目标是确定 R01-GM105993 的结构和动态特性 Ric-8A 和异源三聚体 G 蛋白 Gi (Gαi1) 的 α 亚基之间的复合物。蛋白质调节细胞代谢、分泌、电导率、基因转录、细胞分裂和 G 蛋白的错误调节与细胞运动有关，因此对真核生命至关重要。癌症和一系列与普通医学相关的其他疾病。异三聚体 G 蛋白出现在细胞膜上，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。 Ric-8A 是胚胎发育过程中 Gα 的关键调节因子，Ric-8A 是鸟嘌呤。核苷酸交换因子 (GEF)，通过催化二磷酸鸟苷交换来激活 Gα (GDP) 为 Gα 活性位点的三磷酸鸟苷 (GTP) 该反应的中间体是无核苷酸 Gα:Ric-8A 复合物描述 Ric-8A 结合时发生的结构变化。 Gα·GDP 是了解 Ric-8A 如何激活 Gα 的关键。Ric-8A 也是促进适当的分子伴侣。该提案的两个目标将检验 Gαi1 和可能的 Ric-8A 的假设。对复合体 Gαi1:Ric-8A 中的多种构象状态进行采样，并利用结构信息询问 GEF 和伴侣活动是否在机械上是不可分割的，或者是不同的功能 Ric-8A受蛋白激酶CK2调节的机制也将被解耦。研究目的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，前者处于磷酸化和非磷酸化状态。可变结构域将用作结晶佐剂。作为一种独立的方法来定义 Ric-8A 和 GαI 之间的界面并确定将进行 GEF 和伴侣活性的分析。评估从结构研究中追踪到的残基作为一种或两种介质的作用这些活动将利用小角度 X 射线散射确定 Ric-8A:Gαi1 的整体结构。使用分子动力学和低温电子显微镜对较低分辨率结构进行建模。 Ric-8A和Gαi1的X射线结构的蒙特卡罗模拟将用于检测和识别多个复合物的构象状态。