Structural basis for allosteric regulation of RyR1

RyR1 变构调节的结构基础

• 批准号: 10211076
• 负责人: Oliver Biggs Clarke
• 金额: $ 35.24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211076
• 关键词: Address; Aging; Allosteric Regulation; Allosteric Site; Binding; Binding Proteins; Binding Sites; Calcium; Calcium Binding; Calcium ion; Calmodulin; Cell membrane; Central Core Myopathy; Clinic; Complement; Complex; Coupling; Cresol; Cryoelectron Microscopy; Crystallization; Dantrolene; Data; Development; Disease; Electron Microscopy; Family; Genetic Diseases; Goals; Homeostasis; Human; Ion Channel; Lead; Ligands; Location; Magnesium; Malignant hyperpyrexia due to anesthesia; Maps; Masks; Measures; Mediating; Membrane; Modeling; Molecular; Muscle Contraction; Muscle function; Muscular Atrophy; Mutagenesis; Mutate; Mutation; Myopathy; Peripheral; Physiological; Physiology; Play; Preparation; Process; Proteins; Proteome; Publishing; Regulation; Research; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sampling; Sarcoplasmic Reticulum; Site; Skeletal Muscle; Structure; Tandem Repeat Sequences; Testing; Therapeutic; Tissues; Validation; Variant; Work; base; cryogenics; density; design; experimental study; flexibility; genetic regulatory protein; improved; mutant; particle; receptor; receptor binding; reconstruction; response; scaffold; small molecule; stoichiometry; targeted treatment

PROJECT SUMMARY/ABSTRACT: Ryanodine receptor-mediated calcium release plays an essential role in muscle contraction, and disruption of calcium homeostasis in excitable tissues caused by RyR-mediated calcium leak causes several genetic diseases, as well as contributing to the progressive loss of muscle function that occurs with aging. RyRs are tetrameric ion channels of unusually large size, with each subunit bearing a large cytosolic region that acts as a scaffold for the binding of allosteric modulators, which regulate the channel by binding at sites far from the transmembrane pore. The mechanistic basis of long-range allosteric modulation of RyR1 activity is not well understood. Understanding how allosteric regulators modulate RyR1 will both inform our understanding of RyR in a physiological context, and facilitate the development of molecules that target the channel to treat RyR1 related diseases. The goal of our proposal is to understand the structural mechanism of action of RyR1 allosteric modulators using single particle cryogenic electron microscopy (cryo-EM), complemented by functional analysis. We will investigate calmodulin and dantrolene as examples of long-range allosteric modulators which are endogenous protein binding partners and small molecule ligands, respectively. We will tackle this goal from three directions. In Aim 1, we seek to obtain a complete atomic model of RyR1, including of the peripheral domains where allosteric modulators bind, which have largely eluded sequence assignment due to their flexibility and mobility. We will apply a combination of symmetry expansion and masked refinement to generate a combined reconstruction with improved local resolution in peripheral regions, as well as explore optimization of sample preparation to obtain a more homogeneous particle set. In Aim 2, we will obtain structures of RyR1 in multiple functional states in complex with calmodulin and apocalmodulin, and to use single channel recordings of mutants of both calmodulin and RyR1 to test hypotheses arising from these structures. In Aim 3, we will investigate the mechanism of allosteric inhibition of RyR1 by dantrolene, by first structurally identifying the dantrolene binding site, and then examining the interdependence between inhibition by dantrolene and regulation by calmodulin, magnesium and ATP. Our research will broadly impact the field by unraveling the structural basis of allosteric regulation of an essential ion channel, RyR1. Structural characterization of the dantrolene binding site may lead the way to structure-based design of new RyR1 targeting therapeutics for the treatment of malignant hyperthermia and RyR1-related myopathies.

项目摘要/摘要： ryanodine受体介导的钙释放在肌肉收缩中起着至关重要的作用，并破坏 由RYR介导的钙泄漏引起的激发组织中的钙稳态引起了几种遗传 疾病，以及导致随着衰老而发生的肌肉功能的逐渐丧失。里尔是 异常大小的四聚体离子通道，每个亚基都有一个大的胞质区域，充当一个 用于结合变构调节剂的脚手架，该脚架通过远离位点结合来调节通道 跨膜孔。 RYR1活性的远程变构调制的机械基础不好 理解。了解变构调节器如何调节RYR1都将为我们对RYR的理解提供信息 在生理环境中，并促进针对通道治疗RYR1的分子的发展 相关疾病。我们建议的目的是了解RYR1变构的作用机理 使用单个颗粒低温电子显微镜（Cryo-EM）的调节剂，通过功能分析补充。 我们将调查钙调蛋白和丹特洛琳，作为远程​​变构调节剂的例子 内源性蛋白结合伴侣和小分子配体。我们将从三个目标解决这个目标 方向。在AIM 1中，我们寻求获得RYR1的完整原子模型，包括外围域 变构调节器结合的地方，由于其灵活性和 机动性。我们将使用对称扩展和蒙版改进的组合来产生合并 重建，并改善了外围区域的局部分辨率，并探索样本的优化 准备获得更均匀的粒子集。在AIM 2中，我们将获得RYR1的结构 与钙调蛋白和apocalmodulin的复合物中的功能状态，并使用突变体的单个通道记录 钙调蛋白和RYR1都可以测试由这些结构引起的假设。在AIM 3中，我们将调查 丹特罗烯对ryR1的变构抑制的机制，首先在结构上识别丹托烯结合 站点，然后检查丹特罗来抑制与钙调蛋白调节之间的相互依赖性， 镁和ATP。我们的研究将通过揭开变构的结构基础来广泛影响该领域 基本离子通道RYR1的调节。丹托烯结合位点的结构表征可能会导致 基于结构设计的新RYR1靶向靶向治疗恶性治疗的方法 高温和RYR1相关的肌病。