Structure and dynamics of G protein coupled receptor-G protein complexes

描述（由申请人提供）：本申请的目标是确定G蛋白偶联受体（gpcr）激活特定G蛋白的结构基础。大多数激素和神经递质通过gpcr向细胞传递信息，而gpcr代表了药物开发的最大靶点群。我们的实验室长期致力于阐明gpcr激活G蛋白的结构和机制。在之前的资助期间，我们成功地获得了GPCR-G蛋白复合物的第一个晶体结构：β 2肾上腺素受体(？2AR)与腺苷酸环化酶的刺激蛋白Gs复合物。这种结构为G蛋白激活提供了重要的机制见解，但同时也提出了新的问题，这些问题将在这种竞争性更新中得到解决。具体目标包括：目标1。确定GPCR-G蛋白偶联特异性的结构基础。的结构？2AR-Gs复合体通过GPCR首次提供了跨膜信号的高分辨率见解。然而，需要额外的GPCR-G蛋白复合物来了解G蛋白偶联特异性的结构基础，并确定从？2AR - g结构可推广到其他GPCR-G蛋白对。因此，我们建议获得三种额外的GPCR-G蛋白复合物结构：(1)抗利尿激素受体- gs复合物；(2)结构？2 ar-gi复杂;(3) M2R-Gi配合物的结构。目标2。描述？的形成。2AR-Gs络合物从GDP束缚的Gs异三聚体。的吗?2AR- Gs晶体结构代表了复杂事件循环中的单一状态。络合物形成和解离的过程仍然知之甚少。这些是动态过程，晶体学可能无法解决；然而，？2AR-Gs结构将为设计和解释生物化学和生物物理研究提供基础，以表征复合物的形成和解离机制。在目标2中，我们将描述？2AR和GDP绑定g。这些相互作用可能在G蛋白偶联特异性中起作用。目标3。描述的过程？GTP结合后的2AR -Gs解离。本Aim的目的是了解如何？2AR -Gs复合物在结合GTP后解离成活性信号蛋白，并确定三种成分之间的持续相互作用：？2基于“增大化现实”技术,G ?s和G？目标4。描述G？S螺旋域。最令人惊讶和意想不到的特点？2ar -G结构是构成G？s: ras样GTPase结构域和α螺旋结构域（AHD）。本主题将进一步描述这两个领域之间的相互作用。2AR-Gs复合物以及GTP和GDP约束状态。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the structural basis by which G protein-coupled receptors (GPCRs) activate specific G proteins. The majority of hormones and neurotransmitters communicate information to cells via GPCRs, and GPCRs represent the largest group of targets for drug development. Our laboratories have a long-standing interest in elucidating the structure and mechanism of G protein activation by GPCRs. During the previous funding period we succeeded in obtaining the first crystal structure of a GPCR-G protein complex: the beta2 adrenoceptor (?2AR) in complex with Gs, the stimulatory protein for adenylyl cyclase. This structure provides important mechanistic insight into G protein activation, but at the same time raises new questions that will be addressed in this competitive renewal. Specific Aims include: Aim 1. Determine the structural basis of GPCR-G protein coupling specificity. The structure of the ?2AR-Gs complex provided the first high-resolution insights into transmembrane signaling by a GPCR. However, additional GPCR-G protein complexes will be required to understand the structural basis for G protein coupling specificity, and to determine if the mechanistic insights obtained from the ?2AR -Gs structure are generalizable to other GPCR-G protein pairs. We therefore propose to obtain three additional GPCR-G protein complex structures: (1) the vasopressin receptor-Gs complex; (2) the structure of the ?2AR-Gi complex; and (3) the structure of the M2R-Gi complex. Aim 2. Characterize the formation of the ?2AR-Gs complex from the GDP bound Gs heterotrimer. The ?2AR- Gs crystal structure represents a single state in a complex cycle of events. The process of complex formation and dissociation remains poorly understood. These are dynamic process that may not be addressable by crystallography; however, the ?2AR-Gs structure will provide the basis for designing and interpreting biochemical and biophysical studies to characterize the mechanism of complex formation and dissociation. In Aim 2 we will characterize the low affinity interactions between the ?2AR and GDP bound Gs. These interactions may play a role in G protein coupling specificity. Aim 3. Characterize the process of ?2AR -Gs dissociation following GTP binding. The goal of this Aim is to understand how the ?2AR -Gs complex dissociates into active signaling proteins upon binding GTP and to identify persisting interactions between any of the three components: ?2AR, G?s and G??. Aim 4. Characterize the dynamic behavior of the G?s alpha helical domain. The most surprising and unexpected feature of ?2AR-Gs structures is the flexible link between the two domains that make up G?s: the Ras-like GTPase domain and the alpha helical domain (AHD). This subaim will further characterize the interactions between these two domains in the ?2AR-Gs complex as well as in GTP and GDP bound states.