Structural and dynamic analysis of GRK interaction with G protein-coupled receptors

项目摘要 G蛋白偶联受体激酶(GRKs)磷酸化活化G蛋白偶联受体(GPCRs) 功能是关闭G蛋白信号，开启arrestin介导的信号。GRK有一个模块 将中心催化结构域插入G蛋白信号同源(RH)结构域的调节器中， 其本身由N-末端α-螺旋结构域和C-末端脂质结合区包围。X射线 结晶学表明，RH和催化结构域具有广泛的相互联系和 帮助维持一种不活跃的开放构象的激酶。从机制上洞察GRK监管 在GPCRs中，我们研究了GRK5与β2肾上腺素能受体(β2AR)的相互作用。GRK5一直是 与多种疾病有关，包括心力衰竭和高血压以及GRK5-Q41L基因多态性 在非裔美国人中流行的具有增强的使β2AR脱敏的能力，并防止 发展为充血性心力衰竭。我们已经确定了产生β2AR-GRK5复合体的条件 对这些蛋白质的界面有初步的了解。我们的数据支持一个模型，该模型涉及一个受体- 介导GRK5 RH与受体所必需的催化结构域之间的离子锁的破坏 磷酸化。我们建议利用GRK5与β2AR的相互作用作为模型来进一步剖析 参与GRK激活的机制并深入了解GRK的正常调节和功能 重要的酶家族。 我们的中心假设是，GRK的结构和功能与与 GPCRs。在目标1中，我们将通过诱变来测试我们的初始结构模型，并进一步表征动力学 β2AR-GrK5相互作用的辐射足迹、双电子-电子共振谱和 分子动力学模拟。在目标2中，我们将使用X射线结晶学和单粒子低温电子 显微镜提供对β2AR-Grk5界面的洞察以及β2AR的结构变化 和GRK5结合后发生。我们将确定这些蛋白的结合界面和作用机制 使用分子和生化方法进行激活。在目标3中，我们将重点关注通过以下方式对GRK5进行监管 钙调素。这些研究得到了钙调素-GRK5复合体的晶体结构的支持，该复合体揭示了 钙调蛋白结合破坏RH和催化结构域之间的离子锁定，促进 催化域。我们将通过突变验证钙调蛋白-GRK5的界面，进一步剖析钙调蛋白是如何 激活GRK5并确定钙调蛋白对GRK5在细胞中的定位和功能的作用。总的来说，这些 研究对于理解GRK和GRK-GPCR相互作用具有广泛的意义，并应促进 制定策略，专门调节GRK在疾病治疗中的作用。

Project Summary G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) and function to turn off G protein signaling and turn on arrestin-mediated signaling. GRKs have a modular structure with a central catalytic domain inserted into a regulator of G protein signaling homology (RH) domain, which itself is bracketed by an N-terminal α-helical domain and a C-terminal lipid-binding region. X-ray crystallography has revealed that the RH and catalytic domains have extensive contacts with each other and help to maintain the kinase in an inactive open conformation. To gain mechanistic insight into GRK regulation of GPCRs, we have studied GRK5 interaction with the β2-adrenergic receptor (β2AR). GRK5 has been implicated in several diseases including heart failure and hypertension and a GRK5-Q41L polymorphism prevalent in African Americans has an enhanced ability to desensitize the β2AR and protects against the development of congestive heart failure. We have identified conditions to generate a β2AR-GRK5 complex to gain initial insight on the interface of these proteins. Our data support a model that involves a receptor- mediated disruption of an ionic lock between the GRK5 RH and catalytic domains that is essential for receptor phosphorylation. We propose to utilize GRK5 interaction with the β2AR as a model to further dissect the mechanisms involved in GRK activation and gain insight into the normal regulation and function of this important enzyme family. Our central hypothesis is that GRK structure and function is mechanistically linked to interaction with GPCRs. In aim 1, we will test our initial structural model by mutagenesis and further characterize the dynamics of β2AR-GRK5 interaction using radiolytic footprinting, double electron-electron resonance spectroscopy and molecular dynamics simulations. In aim 2, we will use X-ray crystallography and single particle cryo-electron microscopy to provide insight into the β2AR-GRK5 interface as well as the structural changes that the β2AR and GRK5 undergo upon binding. We will confirm the binding interface of these proteins and the mechanism of activation using molecular and biochemical approaches. In aim 3, we will focus on the regulation of GRK5 by calmodulin. These studies are supported by a crystal structure of a calmodulin-GRK5 complex that reveals that calmodulin binding disrupts the ionic lock between the RH and catalytic domains and promotes closure of the catalytic domain. We will verify the calmodulin-GRK5 interface by mutagenesis, further dissect how calmodulin activates GRK5 and define the role of calmodulin on GRK5 localization and function in cells. Overall, these studies have broad significance for understanding GRKs and GRK-GPCR interaction and should facilitate the development of strategies to specifically regulate GRK function in the treatment of disease.