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

项目概要 该提案旨在阐明三种离子通道的结构和作用机制以及 病毒和细菌的转运体。病原生物利用其膜结合离子通道 和生存的运输者。这些膜蛋白的分子结构信息形成了 合理设计抗病毒和抗生素化合物以对抗和预防病毒和细菌的基础 感染。我们建议 1) 确定 SARS-CoV-2 包膜 (E) 蛋白的结构，其中 组装成刺激宿主炎症小体的阳离子选择性通道； 2）调查 流感M2蛋白的结构机制，形成酸激活的四聚体质子 流感病毒脱衣通道； 3）确定多重耐药细菌的结构 转运蛋白 EmrE，阐明质子耦合底物转运的机制。这些 膜蛋白 – E、M2 和 EmrE – 是遏制 COVID-19 大流行、流感的药物靶标 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 通过检查 B 型流感 M2 (BM2) 突变体来确定 M2 蛋白的传导方向。野生型 (WT) AM2 仅向内传导质子，如转运蛋白，而 WT BM2 双向传导质子，如 规范频道。这种差异与 AM2 进行交替访问的最新数据相关 动作激活，而 BM2 则经历剪刀状动作激活。要了解这些 为了消除差异，我们将研究再现 AM2 内向整流表型的 BM2 突变体。我们会 使用多维固态核磁共振波谱测量其结构和动力学并关联 具有渠道活动的结构信息。在目标 2 中，我们将确定 SARS-CoV-2 E 蛋白的 脂质双层中的跨膜（TM）结构。我们将研究不同阳离子下的E结构 浓度、pH 值和结合抑制剂，以了解 E 如何传导阳离子以及 电导可以被阻止。 2D和3D相关固态NMR实验将在 与通道活动测量相结合。在目标 3 中，我们将研究构象和 通过 31P 和 13C NMR 检测 E 细胞质区域的膜相互作用，以解决 E蛋白的第二个功能的作用是介导病毒出芽和释放。在目标 4 中， 我们将研究 EmrE，它以质子耦合方式流出阳离子药物以产生抗生素 大肠杆菌的耐药性。我们将采用多维19F NMR技术来测量蛋白质药物 距离来限制底物结合口袋的结构。这些研究应该提供 对一些最具破坏性的膜运输机制的详细结构见解 病毒和细菌，并应为改善人类健康的药物设计奠定基础。

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.