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Intro block Structural biology and Biochemistry

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Introduction to the laboratory

        跨膜蛋白是一类包埋其疏水部分于生物膜的蛋白质,主要发挥跨细胞膜的物质运输、能量代谢、信号转导和生物膜系统稳态等多种功能。在人类基因组中,大约30%的基因编码的蛋白质被认为是膜蛋白,而目前在权威蛋白质结构数据库(Protein Databank, PDB)中,膜蛋白结构仅占2%左右,表明对膜蛋白的结构研究相对滞后。然而,大约70% FDA-approved药物的作用靶点是膜蛋白。研究膜蛋白结构可以为以结构为基础的药物设计提供模板。本实验室将利用冷冻电镜,X-射线晶体学等方法研究重要膜蛋白结构,结合生化和电生理以及分子动力学模拟研究膜蛋白行使功能的结构基础及联系。 

        实验室每年招收博士研究生1-3名。热忱欢迎各学科(生物、化学或物理)背景的学生加入。
        同时非常欢迎联合培养研究生来实验室做研究(长期有效)。
        长期招聘博士后,研究助理。

        http://edu.iphy.ac.cn/moreintro.php?id=4381

Nature | 姜道华/赵岩合作揭示囊泡单胺转运体VMAT2的转运及药物抑制的分子机制

2023年12月11日,中国科学院物理研究所/北京凝聚态物理国家研究中心姜道华团队和中国科学院生物物理研究所及赵岩团队合作在Nature上发表了文章 Transport and inhibition mechanisms of human VMAT2,通过冷冻电镜单颗粒技术重构出囊泡单胺转运蛋白VMAT2处于不同构象的高分辨率结构,揭示了VMAT2在运输单胺底物过程中的构象变化及转运机制。

VMAT2分子量仅为56 kDa, 利用冷冻电镜解析如此小的膜蛋白非常困难。为了解决这个难题,研究者通过筛选融合蛋白位点,成功得到性质更加稳定,分子量增大的VMAT2样品用于冷冻透射电镜数据采集,通过计算重构出VMAT2与三种药物分子及底物5羟色胺结合的高分辨率电镜结构。结构分析表明,获得的电镜结构处于胞质朝向、闭塞状态、及囊泡腔朝向的不同构象,代表了VMAT2完整转运循环中的三种典型构象。底物及抑制剂分子结合在 VMAT2的中央结合腔内

该研究为理解VMAT2的底物识别、药物抑制、质子耦合转运过程等分子机制提供了重要的结构基础;为开发靶向VMAT2的构象特异性以及亚型特异性药物提供了重要的结构信息。同时,该研究中解析VMAT2的方法能够应用于其他小型膜蛋白,将促进膜转运蛋白和其他小蛋白的电镜结构解析。

中国科学院物理研究所姜道华特聘研究员和生物物理所赵岩研究员为本文的共同通讯作者。中国科学院物理研究所博士生武迪、生物物理所博士生陈琦浩于卓亚、北京望石智慧黄博、北京大学现代农业研究院赵珺为本文共同第一作者。此外,物理所姜道华组颜芮,生物物理所赵岩组王宇航、苏嘉伟及李娜;望石智慧周峰也为本研究提供了帮助。

原文链接:https://www.nature.com/articles/s41586-023-06926-4

CELL发表首个开放状态Nav1.5结构 | Highlighted by Nature Structure & Molecular Biology news

北京时间2021年9月13日晚23时,中国科学院物理研究所软物质实验室姜道华研究员与美国华盛顿大学William A. Catterall教授郑宁教授合作在顶级杂志《细胞》(Cell在线发表研究论文——《心脏中钠离子通道的开放态结构和门控机制》(Open State Structure and Pore Gating Mechanism of the Cardiac Sodium Channel)。

该研究通过巧妙的设计将钠通道定格在开放状态,并解析了钠通道突变体NaV1.5/QQQ处于开放状态的冷冻电镜结构,揭示了抗心律不齐药物普罗帕酮(Propafenone)与开放状态钠通道的结合位点。结合分子动力学模拟和膜片钳电生理等手段,从原子水平上阐明了钠通道开放状态、钠通道开放状态的药物阻断以及钠通道快速失活的分子机制。

并被Nature Structure & Molecular Biology news & views 专栏报道。

原文如下:

Voltage-gated sodium (NaV) channels mediate the upstroke of the action potential, which requires that they open and close (‘gate’) in response to alterations to the membrane potential within a few milliseconds. The closure of the gates almost immediately after the initial opening, in a process called ‘fast inactivation’, is particularly important to ensure directionality of the action potential and to determine the extent of cellular excitability. Although several cryo-EM structures of human NaV channels have been published so far, all of them were in the highly stable inactivated state, which prevented structural insights into the mechanisms underlying the fast gating

processes.

Writing in Cell, Jiang, Zheng, Catterall and colleagues now report the first structure of an open human NaV(https://doi.org/10.1016/j.cell.2021.08.021), the cardiac channel NaV1.5 that initiates the heartbeat. To obtain channels in the open state, they use a mutant in which the conserved triple-hydrophobic Ile-Phe-Met (IFM) inactivation motif in the intracellular loop that connects domains III and IV is changed to three glutamine residues (QQQ). This alteration is known to completely abolish inactivation, but a steady influx of Na+ is toxic to cells that express the mutant. To overcome this and to purify large amounts of the channel protein, the authors use the antiarrhythmic drug propafenone to block NaV1.5-QQQ while it is being expressed. Subsequent cryo-EM analysis of the sample results in a map resolved to 3.3 Å. The overall structure is very similar to a previously published structure of inactivated NaV1.5, with major conformational changes restricted to the activation gate, the fast-inactivation gate, and the inactivation motif receptor.

Most notably, the hydrophobic pocket,which is occupied by the IFM motif in inactivated NaV structures, is empty in NaV1.5-QQQ, with no detectable cryo-EM density for the region around the QQQ residues, which indicates that these adopt a flexible conformation within the cytoplasm. The displacement of the fast-inactivation motif from its receptor is probably caused by an observed compression of the binding pocket by ~2 Å due to pronounced movements of the S6 segments of domains III and IV. This rearrangement is coupled to dilation of the activation gate (AG), a structure at the intracellular opening of the channel that is connected to the selectivity filter (SF) by a central cavity (CC), to a diameter of approximately 10 Å (left and middle in image). This size is similar to what has been reported before for an open-state structure of the bacterial channel NaVAb. The dimensions of the opening should allow passage of hydrated Na+ through this gate, whose diameter is approximately 7.2 Å. By contrast, the tighter constriction formed by the central SF would require partial dehydration of Na+, ensuring ion selectivity of the channel. Through the use of molecular dynamics simulations,the authors provide evidence that Na+ is able to permeate the putative open state at rates similar to those observed in electrophysiological recordings. The open AG is also wide enough to allow passage of propafenone into the CC, enabling it to reach the antiarrhythmic-drug-receptor site within the open pore, consistent with its open state–specific blocking activity (right in image). The chemistry of the binding of an antiarrhythmic drug is revealed in detail, providing new insights for structure-based drug design.

This new structure now allows mappingof arrhythmia mutations that affect the activation and fast-inactivation gates, gives unprecedented insight into the fast gating processes in NaV channels, and will inform drug design for the targeting of cardiac and other NaV channels.

全文链接:

https://doi.org/10.1016/j.cell.2021.08.021

https://doi.org/10.1038/s41594-021-00672-9