Structural Biology of Rho-Mediated Signaling

Rho GTP酶在心血管系统中介导多种途径，包括对血管生成至关重要的平滑肌钙敏化和细胞迁移现象。该项目的主要目标是确定控制RhoA上游钙敏化途径的分子机制。这些途径涉及一系列核苷酸交换因子激活RhoA GTPase，包括PDZRhogenf和LARG，其中包含将这些蛋白连接到G和丛状蛋白家族受体的结构域，尽管它们直接参与平滑肌生理学尚未得到证实。单个结构域的结构以及这些GEF的多结构域片段将通过核磁共振和X射线结晶学以及其他互补的生物物理方法相结合来确定。通过等温滴定、量热法和噬菌体展示法检测PDZ结构域与网络蛋白C末端结合的功能和机制，以确定相互作用的特异性和亲和力。其战略目标是了解信号转导的膜受体最终如何激活DH-PH结构域串联，该串联催化RhoA上的核苷酸交换。后一种反应的选择性也将通过定点诱变和X射线结晶学进行探索。将与PPG的项目1和3合作，研究GEF的分离结构域和多结构域片段的功能特性以及它们的细胞定位。 该项目还将重点研究丛状蛋白和神经粘连蛋白的结构生物学，它们在许多组织中位于PDZRhogef和LARG的上游，对血管生成至关重要。尽管结构域的边界尚未确定，但丛状蛋白的中心结构域被认为通过一个假定的婴儿床结构域隔离RAC的活性形式。结晶学和/或核磁共振研究将确定这些蛋白质的分子结构以及与RAC的相互作用。最后，还将通过核磁共振和X射线结晶学的协同结合来探索神经粘连蛋白的孤立结构域的结构，以及这些受体的调节机制，以确定结构域间的通信模式。这项工作将为PDZRhogef的激活提供一个模型，并将产生与心血管生物学直接相关的数据。

Rho GTPases mediate diverse pathways in the cardiovascular system, including smooth muscle Ca 2+ sensitization and cell migration phenomena critical for the vasculogenesis. The primary objective of the project is to define the molecular mechanisms that control Ca sensitization pathways upstream of RhoA. These pathways involves activation of the RhoA GTPase by a family of nucleotide exchange factors, including PDZRhoGEF and LARG, which contain domains coupling these proteins to G and receptors of the plexin family, although their direct participation in smooth muscle physiology has not been proven. Structures of the individual domains, as well as of the multi-domain fragments of these GEFs will be determined by a combination of NMR and X-ray crystallography, as well as other complementary biophysical methods. The function and mechanism of the PDZ domain which binds the C-terminus of plexins will be assayed by isothermal titration calorimetry and phage display to determine specificity and affinity of interactions. The strategic aim is to understand how signals transduced membrane receptors ultimately activate the DH-PH domain tandem, which catalyzes the nucleotide exchange on RhoA. The selectivity of the latter reaction will also be probed by site-directed mutagenesis and X, ray crystallography. The functional properties of isolated domains and multi-domain fragments of GEFs, as well as their cellular localization, will be studied in collaboration with Project 1 and 3 within this PPG. The project will also focus on the structural biology of plexins and neuropilins, which function upstream of PDZRhoGEF and LARG in many tissues, and which are of primary importance to vasculogenesis. The central domain of plexin is believed to sequester the active form of Rac through a putative CRIB domain, although the boundaries of structural domains have not been determined. Crystallographic and/or NMR studies will determine the molecular architecture of these proteins and the interaction with Rac. Finally, structures of isolated domains of neuropilins, and the regulatory mechanisms of these receptors will also be probed by a synergistic combination of NMR and X-ray crystallography to determine interdomain communication patterns. This work will provide a model for the PDZRhoGEF activation, and will yield data directly relevant to cardiovascular biology.