CRYSTALLOGRAPHIC STUDIES OF RHOA MEDIATED SIGNALING

RHOA 介导信号传导的晶体学研究

• 批准号: 6642362
• 负责人: Zygmunt S Derewenda
• 金额: $ 18.66万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6642362
• 关键词: X ray crystallography; actins; atherosclerosis; binding sites; bioimaging /biomedical imaging; biological signal transduction; calcium flux; cell migration; crystallization; enzyme activity; enzyme substrate; focal adhesion kinase; geranyl compound; guanine nucleotide exchange factors; guanosinetriphosphatase activating protein; guanosinetriphosphatases; myosins; protein structure function; recombinant proteins; vascular smooth muscle

In this application we propose to study the molecular basis of selected aspects of RhoA-mediated signal transduction in smooth muscle. Specifically, we will study the molecular mechanisms involved in Ca2+ sensitization and migration of vascular smooth muscle cells. These phenomena are key to the contractility in smooth muscle and onset of atherosclerosis. RhoA is a 21 kDa cytosolic GTPase of the Ras-related Rho family of signaling proteins. It shuttles from the biologically active, GTP-bound state in the cell under acute control of numerous pathways. RhoA regulates, among others, cell adhesion and motility, contractile responses, cytokinesis, vascular transport, etc., through the reorganization of the actin skeleton and signaling to myosin. Further, in smooth muscles it is responsible for the effect of Ca2+ sensitization. RhoA plays a critical role in the function of cardiac and vascular smooth muscle. The proposed study will provide a much better understanding of the specific mechanisms by which the signaling pathways function. We have already determined the crystal structure of the RhoA.GDP complex at 2.1 A resolution, and crystals of RhoA with GMPPNP, a non-hydrolyzable GP analogue, have been prepared. Coupled to site-directed mutagenesis and functional studies to be conducted in Project 1, the structural characterization will address the question of what epitopes in RhoA are responsible for downstream signaling to Rho-kinase, and/or effectors, in smooth muscle. Further, we will solve and characterize the structure of a unique GAP, a GTPase activating protein, which is functionally associated with the focal adhesions and shows preference for RhoA and Cdc42Hs as substrates. Crystals for this purpose have been prepared. The structure of the native GAP, as well as the planned characterization of the complexes of GAP with RhoA.GMPPNP and RnoA.GDP.AIF4-, will provide data regarding the activation mechanism and the molecular basis of substrate preference. Finally, we will study the structure-function properties in RhoGDI, a protein that solubilizes RhoA in the cytosol and inhibits the exchanges of the nucleotide. High resolution X-ray crystallography will be used to characterize the details of the geranylgeranyl-binding site of GDI, specifically with respect to solvent structure. Different constructs for crystallization have been prepared and shown to yield crystalline proteins. The ultimate goal is to crystalize the RhoDI-RhoA complex and describe the complete mechanism by which RhoA inhibits nucleotide exchange. To this purpose, we have demonstrated the feasibility of preparing such a complex using recombinant generated in E. coli and yeast.

在此应用中，我们建议研究选定的分子基础 Rhoa介导的平滑肌信号转导的各个方面。 具体而言，我们将研究与Ca2+相关的分子机制 血管平滑肌细胞的致敏和迁移。这些 现象是平滑肌和发作的收缩力的关键 动脉粥样硬化。 Rhoa是与RAS相关的Rho家族的21 kDa胞质GTPase 信号蛋白。它从生物活性，GTP结合的地方穿梭 在众多途径的急性控制下细胞中的状态。 Rhoa 调节细胞粘附和运动性，收缩性 反应，细胞因子，血管运输等 肌动蛋白骨骼和向肌球蛋白发出信号的重组。此外，在 光滑的肌肉是造成Ca2+致敏作用的原因。 Rhoa在心脏和血管平滑的功能中起着至关重要的作用 肌肉。拟议的研究将为您提供更好的了解 信号通路运行的特定机制。我们有 已经确定了2.1 a的rhoa.gdp复合物的晶体结构 分辨率和rhoa的晶体与gmppnp，一种不可用的gp 模拟，已经准备好了。与位置定向的诱变和 在项目1中进行的功能研究，结构性研究 表征将解决Rhoa中什么表现的问题 负责对Rho-kinase和/或效应子的下游信号传导 平滑肌。此外，我们将解决并表征 独特的间隙，一种GTPase激活蛋白，该蛋白在功能上相关 具有焦点粘连，并显示对RhoA和Cdc42hs的偏好为 基材。为此目的已经准备了晶体。结构 天然差距以及复合物的计划表征 与rhoa.gmppnp和rnoa.gdp.aif4-差距的差距将提供有关有关的数据 激活机制和底物偏好的分子基础。 最后，我们将研究rhogdi的结构功能特性 溶解RhoA在细胞质中并抑制交换的蛋白质 核苷酸。高分辨率X射线晶体学将用于 表征了GDI的geranylgeranyl结合位点的细节， 特别是关于溶剂结构。不同的结构 已经准备了结晶并显示出产生结晶 蛋白质。最终目标是使Rhodi-Rhoa综合体和 描述RhoA抑制核苷酸的完整机制 交换。为此，我们证明了 使用大肠杆菌和酵母中产生的重组准备这样的复合物。