Structures and Dynamics of Proton and Cation-Dependent Channels and Transporters

项目摘要 本建议旨在阐明三种离子通道的结构和作用机制。 病毒和细菌的运输者。病原体利用它们的膜结合离子通道 以及生存的运输者。关于这些膜蛋白的分子结构信息形成了 合理设计抗病毒和抗菌化合物以对抗和预防病毒和细菌 感染。我们建议1)确定SARS-CoV-2包膜蛋白(E)的结构，它是 组装成阳离子选择性通道，刺激宿主炎症小体；2)研究 流感M2蛋白形成酸激活四聚体质子的结构机制 流感病毒脱壳的通道；3)确定耐多药细菌的结构 Transporter，Emre，以阐明质子偶联底物运输的机制。这些 膜蛋白-E、M2和EmRE-是抑制新冠肺炎大流行流感的药物靶点 感染和抗生素耐药性。在目标1中，我们将研究质子的结构基础 通过检测B型流感病毒M2(BM2)突变体，确定M2蛋白的传导方向。野生型(WT)AM2 只向内传导质子，就像传送器一样，而WT BM2则像传送器一样双向传导质子 规范通道。这种差异与AM2经历交替访问的最新数据相关 当BM2经历剪刀式运动激活时，运动被激活。要理解这些 不同，我们将研究一个BM2突变体，它概括了AM2的内向纠正表型。我们会 用多维固体核磁共振波谱测量其结构和动力学并关联 与渠道活动相关的结构性信息。在目标2中，我们将确定SARS-CoV-2E蛋白的 脂双层中的跨膜(TM)结构。我们将研究不同阳离子下的E结构 浓度、pH和结合的缓蚀剂，以了解E如何传导阳离子以及E如何 电导可以被阻挡。二维和三维相关固体核磁共振实验将在 与渠道活跃度测量相结合。在目标3中，我们将研究构象和 用~(31)P和~(13)C核磁共振研究E的细胞质区膜相互作用 E蛋白的第二个功能的作用，它介导病毒的萌发和释放。在目标4中， 我们将研究Emre，它以质子偶联的方式流出阳离子药物以产生抗生素 大肠埃希氏菌的耐药性。我们将使用多维~(19)F-核磁共振技术来测量蛋白质-药物 限制衬底结合口袋的结构的距离。这些研究应该提供 对膜运输机制的详细结构洞察在一些最具破坏性的 病毒和细菌，并应为药物设计奠定基础，以改善人类健康。

Project Summary This proposal aims to elucidate the structure and mechanism of action of three ion channels and transporters of viruses and bacteria. Pathogenic organisms use their membrane-bound ion channels and transporters for survival. Molecular structural information about these membrane proteins forms the basis for rational design of antiviral and antibiotic compounds to fight and prevent viral and bacterial infections. We propose to 1) determine the structure of the SARS-CoV-2 envelope (E) protein, which assembles into a cation-selective channel that stimulates the host inflammasome; 2) investigate the structural mechanism of the influenza M2 protein, which forms an acid-activated tetrameric proton channel for influenza virus uncoating; 3) determine the structure of a multidrug-resistant bacterial transporter, EmrE, to elucidate the mechanisms of proton-coupled substrate transport. These membrane proteins – E, M2, and EmrE – are drug targets to curb the COVID-19 pandemic, influenza infections, and antibiotic resistance. In Aim 1 we will investigate the structural basis of the proton conduction direction in M2 proteins by examining an influenza B M2 (BM2) mutant. Wild-type (WT) AM2 conducts protons only inward, like a transporter, while WT BM2 conducts protons bidirectionally, like a canonical channel. This difference is correlated with recent data that AM2 undergoes alternating-access motions to activate while BM2 undergoes a scissor-like motion to activate. To understand these differences, we will study a BM2 mutant that recapitulates the AM2 inward-rectifying phenotype. We will measure its structure and dynamics using multidimensional solid-state NMR spectroscopy and correlate the structural information with channel activities. In Aim 2 we will determine the SARS-CoV-2 E protein’s transmembrane (TM) structure in lipid bilayers. We will investigate the E structures under different cation concentrations, pH and with a bound inhibitor, to understand how E conducts cations and how the conductance can be blocked. 2D and 3D correlation solid-state NMR experiments will be carried out in conjunction with channel activity measurement. In Aim 3 we will investigate the conformation and membrane interaction of the cytoplasmic region of E by 31P and 13C NMR, to address the mechanism of action of the second function of the E protein, which is mediating virus budding and release. In Aim 4, we will investigate EmrE, which effluxes cationic drugs in a proton-coupled manner to cause antibiotic resistance in E. coli. We will employ multidimensional 19F NMR techniques to measure protein-drug distances to constrain the structure of the substrate-binding pocket. These studies should provide detailed structural insights into the mechanism of membrane transport in some of the most devastating viruses and bacteria, and should establish the basis for drug design to improve human health.