Mechanism of G protein Activation by Ric-8A - competitive revision of R01GM105993

 描述(申请人提供)：在R01GM105993的这一竞争性修订中，引入了一个新的目标，即使用驼绒可变重链抗体结构域(纳米体)作为结构支架和功能探针来研究Ric-8A和Gai1之间的相互作用。R01GM105993资助研究，以了解G蛋白是如何被一种名为Ric-8A的蛋白质因子激活的。异三聚体G蛋白调节细胞代谢、分泌、电导、基因转录、细胞分裂和细胞运动，因此对人类所属的真核生物领域的生命是必不可少的。G蛋白的错误调节与癌症和其他一系列与普通医学相关的疾病有关。虽然大多数由异源三聚体G蛋白控制的过程发生在细胞膜上，但最近的研究表明，Gα亚基(GA)也控制细胞质中的某些事件。其中重要的一点是细胞的不对称分裂，这对胚胎发育至关重要。在这一过程中，RIC-8A是GA的关键调节剂。RIC-8A是一种鸟嘌呤核苷酸交换因子，通过在镓的活性中心催化鸟苷二磷酸(GDP)与鸟苷三磷酸(GTP)的交换来激活镓。该反应的中间体是无核苷酸的Ga：RIC-8A络合物。描述当Ric-8A与Ga*GDP结合时发生的结构变化是理解Ric-8A如何激活Ga的关键。RIC-8A也是一种伴侣蛋白，可以促进细胞内GA的正确折叠。R01GM105993的前三个目标解决了Ric-8A：GaI1复合体的结构和动力学行为，以及伴随其形成的Ga和Ric-8A的结构变化。为此，将进行双电子-电子共振(DER)谱、氢-重离子交换-质谱学(HDX-MS)和单分子Förster共振能量转移(SmFRET)实验，以探索GaI-1和Ric-8A结构和动力学的变化。新的目标是利用纳米体来稳定Ric-8A：GaI1络合物的离散结构状态，使其易于结晶和通过X射线结晶学确定结构。改变Ric-8A的全球环境基金或伴侣活性的纳米体将被用来使用HDX-MS鉴定Ric-8A和GA的功能位点。Deer和smFRET将被用来确定削弱或增强Ric-8A活性的纳米体是否减弱或放大全球结构变化或蛋白质动力学。

 DESCRIPTION (provided by applicant): In this competitive revision of R01GM105993, a new aim is introduced to use camelid variable heavy chain antibody domains (nanobodies) as structural scaffolds and functional probes to investigate the interaction between Ric-8A and Gai1. R01GM105993 funds research to understand how G proteins are activated in the cytoplasm of the cell by a protein factor called Ric-8A. Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to life in the domain of eukaryotes to which humans belong. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (Ga) also control certain events in cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of Ga in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that activates Ga by catalyzing the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of Ga. The intermediate in this reaction is the nucleotide-free Ga:Ric-8A complex. Describing the structural changes that occur when Ric-8A binds to Ga*GDP is key to understanding how Ric-8A activates Ga. Ric-8A is also a chaperone that promotes proper folding of Ga in cells. The first three aims of R01GM105993 address the structure and the dynamic behavior of the Ric-8A:Gai1 complex, and the structural changes in Ga and Ric-8A that accompany its formation. In these aims, Double Electron-Electron Resonance (DEER) spectroscopy, Hydrogen-Deuterium eXchange coupled with Mass Spectrometry (HDX-MS) and single molecule Förster Resonance Energy Transfer (smFRET) experiments will be conducted to probe changes in Gai1 and Ric-8A structure and dynamics. The new aim is to use nanobodies to stabilize discrete structural states of the Ric-8A:Gai1 complex to render them amenable to crystallization and structure determination by X-ray crystallography. Nanobodies that alter the GEF or chaperone activity of Ric-8A will be used to identify functional sites in Ric-8A and Ga using HDX-MS. DEER and smFRET will be used to determine whether nanobodies that impair or enhance Ric-8A activity either attenuate or amplify global structural changes or protein dynamics.