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（GRK）磷酸化激活的G蛋白偶联受体（GPCR） 并起到关闭G蛋白信号传导和开启抑制蛋白介导的信号传导的作用。GRK具有模块化 具有插入到G蛋白信号传导同源性（RH）结构域的调节子中的中心催化结构域的结构， 其本身被N-末端α-螺旋结构域和C-末端脂质结合区包围。x射线 晶体学揭示了RH和催化域彼此具有广泛的接触， 有助于将激酶维持在无活性的开放构象。为了获得对GRK调节机制的深入了解 我们研究了GRK 5与β2-肾上腺素能受体（β2-adrenergicreceptor，β2AR）的相互作用。GRK 5已经 与包括心力衰竭和高血压在内的多种疾病有关，GRK 5-Q41 L多态性 在非洲裔美国人中流行的β 2 AR具有增强的脱敏能力，并保护免受 充血性心力衰竭我们已经确定了产生β 2AR-GRK 5复合物的条件， 获得对这些蛋白质界面的初步了解。我们的数据支持一个模型，这个模型涉及一个受体- 介导的GRK 5 RH和催化结构域之间的离子锁的破坏， 磷酸化我们建议利用GRK 5与β 2 AR的相互作用作为模型，以进一步剖析 参与GRK激活的机制，并深入了解这种激活的正常调节和功能。 重要的酶家族 我们的中心假设是，GRK的结构和功能是机械地联系在一起的相互作用， GPCR。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 使用辐解足迹法、双电子-电子共振光谱法和 分子动力学模拟在目标2中，我们将使用X射线晶体学和单粒子低温电子 显微镜，以提供对β 2AR-GRK 5界面以及β2AR 和GRK 5在结合时进行。我们将确定这些蛋白质的结合界面和作用机制。 使用分子和生物化学方法活化。在目标3中，我们将重点关注GRK 5的调节， 钙调素这些研究得到了钙调素-GRK 5复合物的晶体结构的支持，该结构揭示了 钙调蛋白结合破坏RH和催化结构域之间的离子锁，并促进 催化域我们将通过突变来验证钙调素-GRK 5的相互作用，进一步剖析钙调素 激活GRK 5并确定钙调蛋白对细胞中GRK 5定位和功能的作用。总的来说，这些 研究对于理解GRKs和GRK-GPCR相互作用具有广泛的意义， 开发在疾病治疗中特异性调节GRK功能的策略。

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.