STRUCTURAL STUDIES OF RHOA-SPECIFIC GUANINE NUCLEOTIDE EXCHANGE FACTORS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 通过小胞质GT3的信号传导  RhoA位于许多重要信号级联的十字路口，调节平滑肌收缩、神经元形态发生和肌动蛋白细胞骨架重组等过程。RhoA的活性受到两种蛋白质之间复杂相互作用的严格调节  鸟嘌呤核苷酸交换因子（RhoGEF）、GTP酶激活蛋白（RhoGAP）。GEF催化RhoA上鸟嘌呤核苷酸的交换，并因此引起RhoA活化。本研究的目的是表征两种RhoA特异性GEF-白血病相关RhoGEF（LARG）和PDZ-RhoGEF（PRG）的调节机制。假设GEF在非活动状态下通过其四个结构域的相互配置而自我抑制  PDZ、RGSL、DH和PH，并且蛋白质的活化涉及蛋白质的超模块结构的重排。单个GEF结构域的高分辨率结构是已知的，但是没有关于全长蛋白质的结构信息。对这些大分子的研究是困难的，因为预期结构域间连接体的许多部分是非结构化的，因此这些蛋白质不容易结晶。另一方面，它们的尺寸使得几乎不可能使用NMR技术。因此，我们使用小角X射线散射（SAXS），这是越来越多地被认为是一个强大的工具，在溶液中的多结构域蛋白质的表征，探测LARG和PRG的结构，在其自抑制和组成型活性构象，并在复杂的下游效应- RhoA。

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.