2 型兰尼碱受体 RyR2 门控机制的结构基础,Science

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2 型兰尼碱受体 RyR2 门控机制的结构基础
Science ( IF 44.7 ) Pub Date : 2016-09-22 , DOI: 10.1126/science.aah5324
Wei Peng _{1,

2} , Huaizong Shen _{1,

2,

3} , Jianping Wu _{1,

2,

3} , Wenting Guo ₄ , Xiaojing Pan _{1,

2} , Ruiwu Wang ₄ , S. R. Wayne Chen ₄ , Nieng Yan _{1,

2,

3}

Affiliation

对钙通道进行门控 2 型兰尼碱受体 (RyR2) 控制着心肌细胞肌浆网中钙离子的释放——心肌收缩的起始步骤。RyR2 的突变与心脏病有关。彭等人。使用单粒子电子低温显微镜以 4.4 Å 分辨率（钙通道关闭）和 4.2 Å 分辨率（钙通道打开）确定来自猪心脏的 RyR2 的结构。这些结构揭示了域间运动如何导致 RyR2 细胞质区域的构象变化，该变化由中央结构域转导以引起打开或关闭通道的运动。科学，这个问题 p。301 关键细胞内 Ca2+ 通道的细胞质区域的运动由中央结构域转导以门控通道结构域。引言 Ryanodine 受体 (RyRs) 是细胞内 Ca2+ 通道，可控制从肌（内）质网释放 Ca2+。在三种哺乳动物 RyR 异构体中，RyR2 主要在心脏和大脑中表达，并通过称为钙诱导钙释放的机制被 Ca2+ 流入激活。这种 Ca2+ 释放是从肌肉收缩到学习和记忆的细胞过程的基础。RyRs 是已知的最大离子通道，对于同四聚体，其分子量 > 2 兆道尔顿。每个 RyR 原聚体由超过 4500 个残基的细胞质区域和羧基末端跨膜 (TM) 结构域组成。四个相同的 TM 片段包围着一个中央离子传导孔，而细胞质区域则作为与不同配体和蛋白质调节剂相互作用的支架。RyR 通道门控涉及远程变构机制。兔骨骼肌 RyR1 的结构是在 3.8 Å 分辨率下确定的关闭状态和各种较低分辨率的潜在开放状态。阐明 RyR 的门控机制需要以高分辨率对开放状态进行结构测定。基本原理 RyR2 含有超过 150 种与心脏疾病相关的突变，例如儿茶酚胺能多形性 1 型室性心动过速、特发性心室颤动和心源性猝死。RyR2 的结构阐明可能为理解疾病机制和开发潜在的新疗法提供分子基础。我们使用谷胱甘肽 S 转移酶融合的 FKBP12 从猪心脏中纯化内源性 RyR2。为了获得闭合状态下 RyR2 的结构，在整个纯化过程中包含 5 mM EDTA。为了捕获开放的 RyR2，在 20 μM Ca2+ 和可以稳定开放状态的 RyR1 的化合物 2,2',3,5',6-五氯联苯 (PCB95) 的存在下纯化蛋白质。结果 RyR2 的电子显微镜 (EM) 图分别重建为 4.4 和 4.2 Å 分辨率的闭合和打开状态。与 RyR1 的结构相比，螺旋结构域 2 C 末端的许多犰狳重复在 RyR2 中是不可见的，这可能是由于内在的灵活性。在 20 μM Ca2+ 下，封闭结构中的细胞质门扩大约 8 Å，导致收缩位点从 Ile4868 转移到 Gln4864。四个 Gln4864 残基包围了一个直径约为 4 Å 的门，允许 Ca2+ 在单个文件中通过。结论开放和封闭 RyR2 的结构比较显示含有犰狳的细胞质结构域（包括氨基末端结构域、手柄结构域和螺旋结构域）的很少结构域内重排。这些域之间的相对偏移导致细胞质冠层外围的呼吸运动和中央域的旋转。马蹄形中央结构域，其凸面与三个犰狳结构域相互作用，其凹面环绕通道结构域的细胞质 O 形环，作为主要传感器，将细胞质结构域的构象变化整合并转化为通道选通。然而，Ca2+ 感应和激活 RyR2 的机制仍有待阐明。来自猪心脏的 RyR2 在接近原子分辨率下处于闭合和打开状态的低温 EM 结构。（上）封闭和开放 RyR2 电子显微照片的代表性二维类平均值。（下）这两种结构相对于跨膜域叠加。蓝色箭头表示细胞质区域从关闭状态到打开状态的整体转变。SR，肌浆网。RyR2 是一种高电导细胞内钙 (Ca2+) 通道，可控制多种细胞的肌（内）质网释放 Ca2+。在这里，我们报告了使用单粒子电子低温显微镜确定的近原子分辨率处于打开和关闭状态的猪心脏 RyR2 的结构。结构比较显示整个细胞质区域的呼吸运动是由氨基末端结构域 (NTD)、螺旋结构域和手柄结构域的域间运动引起的，而在这些含有犰狳重复序列的结构域中几乎没有观察到结构域内的变化。中央结构域的向外旋转整合了细胞质区域的构象变化，通过耦合运动导致细胞质门的扩张。我们的结构和突变特征为 RyR 的门控和疾病机制提供了重要的见解。它整合了细胞质区域的构象变化，通过耦合运动导致细胞质门的扩张。我们的结构和突变特征为 RyR 的门控和疾病机制提供了重要的见解。它整合了细胞质区域的构象变化，通过耦合运动导致细胞质门的扩张。我们的结构和突变特征为 RyR 的门控和疾病机制提供了重要的见解。

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Structural basis for the gating mechanism of the type 2 ryanodine receptor RyR2

Gating a calcium channel The type 2 ryanodine receptor (RyR2) controls the release of calcium ions from the sarcoplasmic reticulum in cardiac cells—the initiating step in cardiac muscle contraction. Mutations in RyR2 are associated with cardiac diseases. Peng et al. used single-particle electron cryomicroscopy to determine the structure of RyR2 from porcine heart at 4.4-Å resolution with the calcium channel closed and at 4.2-Å resolution with the calcium channel open. The structures reveal how interdomain motions result in a conformational change in the cytoplasmic region of RyR2 that is transduced by a central domain to cause motions that open or close the channel. Science, this issue p. 301 Motion of the cytoplasmic region of a key intracellular Ca2+ channel is transduced by a central domain to gate the channel domain. INTRODUCTION Ryanodine receptors (RyRs) are intracellular Ca2+ channels that control the release of Ca2+ from the sarco(endo)plasmic reticulum. Among the three mammalian RyR isoforms, RyR2 is primarily expressed in the heart and brain and is activated by Ca2+ influx by a mechanism known as calcium-induced calcium release. This Ca2+ release is fundamental to cellular processes ranging from muscle contraction to learning and memory. RyRs are the largest known ion channels with a molecular mass of > 2 megadalton for a homotetramer. Each RyR protomer consists of a cytoplasmic region of over 4500 residues and a carboxyl terminal transmembrane (TM) domain. The four identical TM segments enclose a central ion-conducting pore, whereas the cytoplasmic regions serve as a scaffold for interactions with diverse ligands and protein modulators. RyR channel gating involves a long-range allosteric mechanism. The structures of rabbit skeletal muscle RyR1 were determined at 3.8-Å resolution for the closed state and various lower resolutions for the potentially open states. Elucidating the gating mechanism of RyR requires structural determination of the open states at high resolution. RATIONALE RyR2 harbors more than 150 mutations associated with cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia type 1, idiopathic ventricular fibrillation, and sudden cardiac death. Structural elucidation of RyR2 may provide the molecular basis for understanding disease mechanisms and for the development of potential novel therapeutics. We purified endogenous RyR2 from porcine heart using glutathione S-transferase–fused FKBP12. To obtain the structure of RyR2 in the closed state, 5 mM EDTA was included throughout purification. To capture an open RyR2, the protein was purified in the presence of 20 μM Ca2+ and the compound 2,2′,3,5′,6-pentachlorobiphenyl (PCB95) that can stabilize RyR1 in the open state. RESULTS The electron microscopy (EM) maps for RyR2 were reconstructed to 4.4- and 4.2-Å resolutions for the closed and open states, respectively. Compared to the structure of RyR1, a number of armadillo repeats in the C terminus of the helical domain 2 were invisible in RyR2, likely due to intrinsic flexibility. At 20 μM Ca2+, the cytoplasmic gate in the closed structure is dilated by approximately 8 Å, resulting in the shift of the constriction site from Ile4868 to Gln4864. The four Gln4864 residues enclose a gate with a diameter of approximately 4 Å, allowing Ca2+ passage in a single file. CONCLUSION Structure comparison of the open and closed RyR2 shows little intradomain rearrangement of the armadillo-containing cytoplasmic domains including the amino terminal domain, the Handle domain, and the Helical domain. Relative shifts between these domains result in the breathing motion of the periphery of the cytoplasmic canopy and the rotation of the Central domain. The horseshoe-shaped Central domain, with its convex side interacting with the three armadillo domains and its concave side wrapping around the cytoplasmic O-ring of the channel domain, serves as the primary transducer that integrates and translates the conformational changes of the cytoplasmic domains to channel gating. However, the mechanism of Ca2+ sensing and activation of RyR2 remains to be elucidated. Cryogenic EM structures of RyR2 from porcine heart in both the closed and open states at near-atomic resolutions. (Top) Representative two-dimensional class averages of electron micrographs of the closed and open RyR2. (Bottom) The two structures are superimposed relative to the transmembrane domain. The blue arrows indicate the overall shifts of the cytoplasmic region from the closed state to the open state. SR, sarcoplasmic reticulum. RyR2 is a high-conductance intracellular calcium (Ca2+) channel that controls the release of Ca2+ from the sarco(endo)plasmic reticulum of a variety of cells. Here, we report the structures of RyR2 from porcine heart in both the open and closed states at near-atomic resolutions determined using single-particle electron cryomicroscopy. Structural comparison reveals a breathing motion of the overall cytoplasmic region resulted from the interdomain movements of amino-terminal domains (NTDs), Helical domains, and Handle domains, whereas almost no intradomain shifts are observed in these armadillo repeats–containing domains. Outward rotations of the Central domains, which integrate the conformational changes of the cytoplasmic region, lead to the dilation of the cytoplasmic gate through coupled motions. Our structural and mutational characterizations provide important insights into the gating and disease mechanism of RyRs.

更新日期：2016-09-22

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