CRYSTALLOGRAPHIC STUDIES OF RHOA MEDIATED SIGNALING

在本申请中，我们建议研究选择的 平滑肌中RhoA介导的信号转导方面。 具体来说，我们将研究Ca 2+参与的分子机制， 血管平滑肌细胞的致敏和迁移。这些 这种现象是平滑肌收缩性和 动脉粥样硬化 RhoA是Ras相关的Rho家族的21 kDa胞质GT3， 信号蛋白它从生物活性的GTP结合的 在细胞中处于许多途径的急性控制下。RhoA 调节细胞粘附和运动性、收缩 反应、胞质分裂、血管运输等，通过 肌动蛋白骨架的重组和肌球蛋白的信号传导。进一步 平滑肌中，它负责Ca 2+敏化的作用。 RhoA在心脏和血管平滑肌的功能中起着关键作用， 肌肉.拟议的研究将使人们更好地了解 信号传导通路发挥作用的特定机制。我们有 已经确定了RhoA.GDP复合物的晶体结构， 分辨率，以及RhoA与GMPPNP（一种不可水解的GP）的晶体 类似物，已经准备好了。结合定点诱变， 功能研究将在项目1中进行， 表征将解决RhoA中的表位是什么的问题 负责Rho激酶和/或效应子的下游信号传导， 平滑肌此外，我们将解决和表征的结构， 一种独特的GAP，一种GT3激活蛋白，在功能上与 与局灶性粘连，并显示出对RhoA和Cdc 42 Hs的偏好， 印刷受体.已经制备了用于此目的的晶体。的结构 天然GAP，以及复合物的计划表征 GAP与RhoA.GMPPNP和RnoA.GDP.AIF4-，将提供有关 活化机制和底物选择性的分子基础。 最后，我们将研究RhoGDI的结构-功能特性， 一种在胞质溶胶中溶解RhoA并抑制 核苷酸。高分辨率X射线晶体学将用于 表征GDI的香叶基香叶基结合位点的细节， 特别是关于溶剂结构。不同的结构， 已经制备了结晶，并显示出产生结晶 proteins.最终目标是使RhoDI-RhoA复合物结晶， 描述了RhoA抑制核苷酸的完整机制 交易所为此，我们证明了 使用在E.大肠杆菌和酵母菌。

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.