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

GRK 与 G 蛋白偶联受体相互作用的结构和动态分析

• 批准号: 9913308
• 负责人: Jeffrey L Benovic
• 金额: $ 52.91万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913308
• 关键词: ADRBK1 gene; African American; Agonist; Architecture; Arrestins; Binding; Biochemical; Biological; Biophysics; C-terminal; Calmodulin; Cardiovascular Diseases; Catalytic Domain; Cell Nucleus; Cell membrane; Cells; Complement; Complex; Congenital Heart Defects; Congestive Heart Failure; Coupled; Cryoelectron Microscopy; Crystallization; Data; Deuterium; Development; Disease; Docking; Electron Microscopy; Electrons; Enzymes; Family; G protein coupled receptor kinase; G-Protein-Coupled Receptors; G-substrate; GRK1 gene; GRK5 gene; GRK6 gene; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genetic Polymorphism; Heart Hypertrophy; Heart failure; Human; Hydrogen; Hypertension; Ligands; Link; Lipid Binding; Lobe; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Metabolic Diseases; Mission; Modeling; Molecular; Molecular Conformation; Mutagenesis; N-terminal; Normal Cell; Organism; Phospholipids; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protein Analysis; Proteins; Public Health; Regulation; Research; Resolution; Role; Signal Transduction; Signaling Protein; Site; Spectrum Analysis; Structural Models; Structure; Surface; Testing; United States National Institutes of Health; X-Ray Crystallography; beta-2 Adrenergic Receptors; cardiogenesis; crosslink; desensitization; human disease; insight; molecular dynamics; nanobodies; nervous system disorder; particle; protein activation; public health relevance; receptor; receptor binding; receptor function

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.

项目概要 G 蛋白偶联受体激酶 (GRK) 磷酸化激活的 G 蛋白偶联受体 (GPCR) 并发挥关闭 G 蛋白信号传导并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 G 蛋白信号同源 (RH) 结构域调节器的中央催化结构域的结构， 它本身被 N 端 α 螺旋结构域和 C 端脂质结合区包围。 X射线 晶体学表明 RH 和催化域彼此有广泛的接触并且 有助于维持激酶处于非活性开放构象。深入了解 GRK 调节的机制 GPCR 中，我们研究了 GRK5 与 β2 肾上腺素受体 (β2AR) 的相互作用。 GRK5 已 与多种疾病有关，包括心力衰竭和高血压以及 GRK5-Q41L 多态性 非洲裔美国人中流行的β2AR 具有增强的脱敏能力并可防止 充血性心力衰竭的发展。我们已经确定了生成 β2AR-GRK5 复合物的条件 初步了解这些蛋白质的界面。我们的数据支持涉及受体的模型- 介导 GRK5 RH 和催化结构域之间离子锁的破坏，这对受体至关重要 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其正常调节和功能 重要的酶家族。 我们的中心假设是 GRK 结构和功能在机制上与 GPCR。在目标 1 中，我们将通过诱变测试我们的初始结构模型并进一步表征动力学 使用放射分解足迹、双电子-电子共振光谱和 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冷冻电子 显微镜可深入了解 β2AR-GRK5 界面以及 β2AR 的结构变化 和GRK5结合后发生。我们将确认这些蛋白质的结合界面及其机制 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 GRK5 的监管 钙调蛋白。这些研究得到钙调蛋白-GRK5 复合物晶体结构的支持，该复合物揭示了 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。我们将通过诱变验证钙调蛋白-GRK5接口，进一步剖析钙调蛋白如何 激活 GRK5 并定义钙调蛋白对 GRK5 定位和细胞功能的作用。总体而言，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 制定在疾病治疗中特异性调节 GRK 功能的策略。