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

项目摘要 该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 和运输工具来生存这些膜蛋白的分子结构信息构成了 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， 组装成刺激宿主炎性小体的阳离子选择性通道; 2）研究 流感M2蛋白的结构机制，形成酸活化的四聚体质子 流感病毒脱壳通道; 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， 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 通过检查流感B M2（BM 2）突变体确定M2蛋白的传导方向。野生型（WT）AM 2 只向内传导质子，就像一个转运体，而WT BM 2双向传导质子，就像一个 Canonical频道这种差异与AM 2经历交替访问的最新数据相关 运动来激活，而BM 2经历剪刀状运动来激活。理解这些 差异，我们将研究一个BM 2突变体，重演AM 2内向整流表型。我们将 使用多维固态NMR光谱测量其结构和动力学， 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 电导可以被阻断。2D和3D相关固态NMR实验将在 结合信道活动测量。在目标3中，我们将研究构象和 通过31 P和13 C NMR分析E的胞质区域的膜相互作用，以解决 E蛋白的第二个功能，即介导病毒出芽和释放的作用。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， E.杆菌我们将采用多维19 F NMR技术来测量蛋白质-药物 距离，以限制底物结合口袋的结构。这些研究将提供 在一些最具破坏性的， 病毒和细菌，并应建立药物设计的基础，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.