STRUCTURAL STUDIES OF RHOA-SPECIFIC GUANINE NUCLEOTIDE EXCHANGE FACTORS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞内小分子GTP酶RhoA介导的信号转导是许多重要的信号转导通路的交叉点，它调节着许多重要的信号转导过程，如平滑肌收缩、神经元形态发生和肌动蛋白细胞骨架的重组。RhoA的活性受到两个蛋白质之间复杂的相互作用的严格调控，这两个蛋白质是鸟嘌呤核苷酸交换因子(Rhogef)和GTP酶激活蛋白(RhoGAP)。GEFS催化鸟嘌呤核苷酸在RhoA上的交换，从而引起RhoA的激活。本研究的目的是研究两种RhoA特异性GEF的调控机制-白血病相关的Rhogef(LARG)和PDZ-Rhogef(PRG)。假设处于非活动状态的GEF是通过其四个结构域PDZ、RGSL、DH和PH的相互配置而被自动抑制的，并且蛋白质的激活涉及蛋白质的超模块结构的重排。虽然已知单个环境基金结构域的高分辨结构，但关于全长蛋白质的结构信息尚不清楚。对这些大分子的研究是困难的，因为结构域间连接物的许多部分被认为是非结构化的，因此这些蛋白质不利于结晶。另一方面，它们的尺寸使得使用核磁共振技术几乎是不可能的。因此，我们使用小角X射线散射(SAXS)，这是越来越被认为是表征溶液中多结构域蛋白质的有力工具，来探索LARG和PRG在其自抑制构象和结构活性构象中的结构，以及与其下游效应元件RhoA的复合体的结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Signaling via small cytosolic GTPase  RhoA lies at the crossroads of many important signaling cascades, regulating processes such as smooth muscle contraction, neuronal morphogenesis, and reorganization of actin cytoskeleton. Activity of RhoA is tightly regulated by a sophisticated interplay between two proteins  guanine nucleotide exchange factor (RhoGEF), GTPase-activating protein (RhoGAP). GEFs catalyze the exchange of a guanine nucleotide on RhoA, and consequently cause RhoA activation. The aim of this research is to characterize the mechanism of regulation of two RhoA-specific GEFs - leukemia-associated RhoGEF (LARG) and PDZ-RhoGEF (PRG). It is assumed that GEFs in their inactive state are autoinhibited by the mutual disposition of their four domains  PDZ, RGSL, DH, and PH, and that the activation of the protein involves rearrangements of proteins` supramodular architecture. The high resolution structures of individual GEF domains are known, however there is no structural information about the full-length protein. Studies of these large molecules are difficult, because many portions of interdomain linkers are expected to be unstructured, and therefore such proteins are recalcitrant to crystallization. On the other hand, their size makes it virtually impossible to use NMR techniques. We therefore used small-angle X-ray scattering (SAXS), which is increasingly recognized as a powerful tool in the characterization of multidomain proteins in solution, to probe the structure of LARG and PRG in their autoinhibited and constitutively active conformations, and in complex with their downstream effector - RhoA.