Structural Biology of Rho-Mediated Signaling

Rho GTP酶在心血管系统中介导多种途径，包括平滑肌Ca 2+增敏和血管发生中的细胞迁移现象。该项目的主要目标是确定控制RhoA上游钙致敏途径的分子机制。这些途径涉及通过核苷酸交换因子家族激活RhoA GTdR，包括PDZRhoGEF和LARG，其含有将这些蛋白质偶联至丛蛋白家族的G和受体的结构域，尽管它们直接参与平滑肌生理学尚未得到证实。这些GEF的各个结构域以及多结构域片段的结构将通过NMR和X射线晶体学以及其他互补生物物理方法的组合来确定。结合丛蛋白C-末端的PDZ结构域的功能和机制将通过等温滴定量热法和噬菌体展示来测定相互作用的特异性和亲和力。战略目标是了解信号转导的膜受体如何最终激活DH-PH结构域串联，其催化RhoA上的核苷酸交换。后一反应的选择性也将通过定点诱变和X射线晶体学来探测。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，将与本PPG中的项目1和3合作研究。 该项目还将侧重于plexins和neuropilins的结构生物学，它们在许多组织中的PDZRhoGEF和LARG上游发挥作用，并且对血管生成至关重要。据信丛蛋白的中心结构域通过推定的CRIB结构域隔离活性形式的Rac，尽管结构域的边界尚未确定。晶体学和/或NMR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，神经纤毛蛋白的隔离域的结构，以及这些受体的调节机制也将通过NMR和X-射线晶体学的协同组合来探测，以确定域间通信模式。这项工作将为PDZRhoGEF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.