CRYSTALLOGRAPHIC STUDIES OF RHOA MEDIATED SIGNALING

在此应用中，我们建议研究所选的分子基础 平滑肌中 RhoA 介导的信号转导的各个方面。 具体来说，我们将研究Ca2+所涉及的分子机制。 血管平滑肌细胞的敏化和迁移。这些 现象是平滑肌收缩性和开始的关键 动脉粥样硬化。 RhoA 是 Ras 相关 Rho 家族的 21 kDa 胞质 GTP 酶 信号蛋白。它从具有生物活性、GTP 结合的 细胞中的状态受到许多途径的严格控制。罗阿 除其他外，调节细胞粘附和运动、收缩 反应、胞质分裂、血管运输等 肌动蛋白骨架的重组和肌球蛋白的信号传导。进一步地，在 平滑肌负责 Ca2 敏化作用。 RhoA在心脏和血管平滑肌功能中起着至关重要的作用 肌肉。拟议的研究将提供更好的理解 信号通路发挥作用的具体机制。我们有 已经确定了 2.1 A 下 RhoA.GDP 复合物的晶体结构 分辨率，以及带有 GMPPNP（一种不可水解的 GP）的 RhoA 晶体 类似物，已准备好。结合定点诱变和 项目 1 进行功能研究，结构 表征将解决 RhoA 中的表位是什么的问题 负责 Rho 激酶和/或效应器的下游信号传导， 平滑肌。此外，我们将解决并表征一个结构 独特的 GAP，一种 GTP 酶激活蛋白，与功能相关 与粘着斑并显示出对 RhoA 和 Cdc42Hs 的偏好 基材。为此目的已经制备了晶体。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，RhoGDI 是一个 溶解细胞质中 RhoA 并抑制 RhoA 交换的蛋白质 核苷酸。高分辨率 X 射线晶体学将用于 表征 GDI 香叶基香叶基结合位点的细节， 特别是关于溶剂结构。不同的构造 已制备结晶并显示可产生结晶 蛋白质。最终目标是结晶 RhoDI-RhoA 复合物并 描述 RhoA 抑制核苷酸的完整机制 交换。为此，我们论证了以下方案的可行性： 使用大肠杆菌和酵母中产生的重组体制备这样的复合物。

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.