Structural Analysis of the Rhodopsin-Transducin Complex

描述（由申请人提供）：G蛋白偶联受体（GPCR）是一大组7跨膜螺旋受体，需要配体依赖性激活以启动异源三聚体（a，B，y）G蛋白介导的细胞内信号级联。由于其生理相关性和药理学易处理性，GPCR是许多药物发现工作的焦点。G蛋白通过其激动剂刺激的GPCR（R*）的活化需要结构信号从受体结合界面传播到G蛋白的鸟嘌呤核苷酸结合口袋。GPCR与其同源G蛋白相互作用的结构基础以及随后通过R* 激活G蛋白的结构基础尚未完全理解。本研究的总体目标是开发和应用高分辨率核磁共振（NMR）方法来探测从R* 到G蛋白的结构信号传播的结构基础，特别关注阐明导致鸟嘌呤核苷酸交换的a亚基的结构变化。对于这些研究，将使用光激活的GPCR、视紫红质对G蛋白、转导素（Gt）的信号传导作为模型系统。Gt与溶解的天然和突变的视紫红质，以及R* 的可溶性模拟物的相互作用，将被用来允许观察和捕获伴随信号传递的离散状态。使用同位素标记的G-蛋白a-亚基，异源NMR方法将用于选择性地映射该亚基在与未标记的By-亚基形成异源三聚体时以及当被困在离散的R* 结合状态时的结构变化。围绕R* 介导的信号传递机制的3个基本结构问题将得到解决：1。当G蛋白异源三聚体与R* 相互作用时，a亚基的受体结合界面发生了什么结构变化？2. G蛋白异源三聚体与R* 相互作用时，鸟嘌呤核苷酸结合位点的结构发生了什么变化？3.由α-亚基与R* 的结合界面相互作用引起的结构变化如何与鸟嘌呤核苷酸结合位点的构象变化相关？我们希望这项工作将对我们理解R* 激活G蛋白的机制产生根本性影响，并为理解与视觉功能障碍相关的天然视紫红质和Gt突变的结构后果奠定基础。

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are a large group of 7 transmembrane helix receptors that require ligand-dependent activation to initiate heterotrimeric (a, B, y) G-protein mediated intracellular signaling cascades. Due to their physiological relevance and pharmacological tractability, GPCRs are the focus of numerous drug discovery efforts. Activation of a G-protein by its agonist stimulated GPCR (R*) requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide-binding pocket of the G-protein. The structural basis for the interaction of a GPCR with its cognate G-protein, and the subsequent activation of the G-protein by R*, are not fully understood. The overall goal of this research is to develop and apply high-resolution nuclear magnetic resonance (NMR) methods to probe the structural basis for the propagation of structural signals from R* to the G-protein, with a specific focus on elucidating structural changes in the a-subunit that lead to guanine nucleotide exchange. For these studies, signaling of the G-protein, transducin (Gt), by the light-activated GPCR, rhodopsin, will be used as a model system. The interaction of Gt with solubilized native and mutant rhodopsins, as well as soluble mimics of R*, will be employed to allow for the observation and trapping of discrete states accompanying signal transfer. Using isotope-labeled G-protein a-subunits, heteronuclear NMR methods will be used to selectively map structural changes in this subunit upon heterotrimer formation with unlabeled By-subunits and when trapped in discrete R* bound states. 3 fundamental structural questions surrounding the mechanism of R* mediated signal transfer will be addressed: 1. What structural changes in the receptor binding interface of the a-subunit occur upon interaction of the G-protein heterotrimer with R*?; 2. What are the structural changes in the guanine nucleotide binding site that occur upon interaction of the G-protein heterotrimer with R*?; 3. How are structural changes that result from the interaction of the binding interface of the a-subunit with R* correlated to changes in the conformation of the guanine nucleotide binding site? We expect that this work will have a fundamental impact on our understanding of the mechanisms governing activation of G-proteins by R* and form a basis for understanding the structural consequences of naturally occurring rhodopsin and Gt mutations associated with visual dysfunction.