Structural basis for allosteric regulation of RyR1

项目总结/摘要： Ryanodine受体介导的钙释放在肌肉收缩中起着重要作用，并且破坏了肌肉的收缩。 由RyR介导的钙渗漏引起的可兴奋组织中的钙稳态引起几种遗传性的 疾病，以及有助于随着年龄的增长而发生的肌肉功能的逐渐丧失。RyR是 四聚体离子通道的尺寸异常大，每个亚基带有一个大的胞质区域，作为一个 支架用于结合变构调节剂，其通过在远离通道的位点结合来调节通道。 跨膜孔RyR 1活性的远程变构调节的机制基础尚不清楚 明白了解变构调节剂如何调节RyR 1将有助于我们了解RyR 并促进靶向通道以治疗RyR 1的分子的发展 相关疾病。我们建议的目标是了解RyR 1变构作用的结构机制， 使用单粒子低温电子显微镜（cryo-EM），辅以功能分析，对调制器进行分析。 我们将研究钙调素和丹曲林作为远程变构调节剂的例子， 内源性蛋白结合配偶体和小分子配体。我们将从三个方面着手实现这一目标 方向在目标1中，我们试图获得一个完整的RyR 1原子模型，包括周边结构域 其中变构调节剂结合，其由于其灵活性而在很大程度上回避了序列分配， 迁移率我们将应用对称扩展和掩码细化的组合来生成组合的 在周边区域提高局部分辨率重建，以及探索样本的优化 制备以获得更均匀的颗粒集。在目标2中，我们将获得多个RyR 1的结构， 与钙调素和脱钙调素复合的功能状态，并使用突变体的单通道记录 的钙调素和RyR 1来测试这些结构所产生的假设。在目标3中，我们将研究 丹曲林对RyR 1的变构抑制机制，通过首先在结构上鉴定丹曲林结合 站点，然后检查丹曲林抑制和钙调素调节之间的相互依赖性， 镁和ATP。我们的研究将通过揭示变构的结构基础来广泛影响该领域。 一个重要的离子通道，RyR 1的调节。丹曲林结合位点的结构表征可能导致 基于结构设计新型RyR 1靶向治疗恶性肿瘤的方法 高热和RyR 1相关肌病。

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.