CRYSTALLOGRAPHIC STUDIES OF RHOA MEDIATED SIGNALING

在这一应用中，我们建议研究选定的分子基础 RhoA介导的平滑肌信号转导方面。 具体地说，我们将研究钙离子的分子机制。 血管平滑肌细胞的敏化和迁移。这些 现象是平滑肌收缩和血管紧张性改变的关键 动脉硬化。 RhoA是Ras相关的Rho家族的21 kDa胞浆GTP酶。 信号蛋白。它穿梭于生物活性的、与GTP结合的 在许多通路的急性控制下，细胞处于一种状态。RhoA 除其他外，调节细胞黏附和运动性，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对肌球蛋白的信号传递。此外，在 平滑肌肉，它负责钙的敏化作用。 RhoA在心脏和血管通畅功能中起关键作用 肌肉。拟议的研究将使人们更好地了解 信号通路发挥作用的特定机制。我们有 已经测定了2.1A的RhoA·GDP络合物的晶体结构 RhoA与非水解性GP GMPPNP的拆分和结晶 类似的，已经准备好了。与定点突变相结合， 将在工程项目1中进行的功能研究 表征将解决RhoA中的表位是什么的问题 负责Rho-Kinase和/或效应器的下游信号传递 平滑的肌肉。此外，我们还将解决并刻画 独特的GAP，一种GTP酶激活蛋白，在功能上与 与焦点粘连，并表现出对RhoA和Cdc42Hs的偏好 底物。用于此目的的晶体已被制备出来。的结构 原生缺口，以及复合体的计划表征 与RhoA.GMPPNP和RnoA.GDP.AIF4-的差距将提供以下数据 底物选择性的激活机制和分子基础。 最后，我们将研究RhoGDI的结构-功能性质，一个 一种使RhoA溶解在胞浆中并抑制RhoA交换的蛋白质 核苷酸。高分辨率X射线结晶学将用于 表征GDI的香叶基香叶基结合部位的细节， 特别是在溶剂结构方面。不同的构造 已经制备了结晶，并被证明能产生结晶 蛋白质。最终目标是使Rhodi-RhoA复合体具体化 描述RhoA抑制核苷酸的完整机制 交换。为此，我们论证了 利用在大肠杆菌和酵母中产生的重组制备这样的复合体。

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.