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

质子和阳离子依赖性通道和转运蛋白的结构和动力学

• 批准号: 10659039
• 负责人: Mei Hong
• 金额: $ 30.39万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659039
• 关键词: 2019-nCoV; 3-Dimensional; ABCB1 gene; Acids; Address; Adopted; Affect; Amiloride; Amino Acid Sequence; Antibiotic Resistance; Antibiotics; Antiviral Agents; Bacteria; Bacterial Infections; Binding; Binding Sites; Biological Assay; COVID-19 pandemic; Cations; Cells; Chemicals; Coupled; Couples; Cytoplasm; Cytoplasmic Tail; Data; Dehydration; Dimensions; Drug Design; Drug Efflux; Drug Targeting; E protein; Endoplasmic Reticulum; Escherichia coli; Goals; Golgi Apparatus; H19 gene; Health; Human; Hydrophobic Interactions; Infection; Inflammasome; Influenza; Influenza B Virus; Integral Membrane Protein; Ion Channel; Knowledge; Label; Length; Lipid Bilayers; Lipids; Literature; M2 protein; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Molecular; Molecular Conformation; Motion; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; NMR Spectroscopy; Organism; Pathogenicity; Pharmaceutical Preparations; Phenotype; Protein Dynamics; Proteins; Protons; Public Health; Recording of previous events; Research; Resistance to infection; Resolution; Spanish flu; Structure; Techniques; Time; Transmembrane Domain; Transmembrane Transport; Vertebral column; Viral; Viral Proteins; Virus; Virus Diseases; antiviral drug development; bacterial resistance; combat; design; env Gene Products; experimental study; fighting; improved; influenza M2; influenza infection; influenzavirus; inhibitor; insight; membrane model; mimetics; mutant; nanometer; novel; prevent; protonation; rational design; seasonal influenza; solid state nuclear magnetic resonance

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.

项目摘要 该提案旨在阐明三个离子通道的作用结构和机制， 病毒和细菌的转运蛋白。致病生物使用其膜结合的离子通道 和转运蛋白生存。有关这些膜蛋白的分子结构信息形成 抗病毒和抗生素化合物的合理设计基础，以战斗和预防病毒和细菌 感染。我们建议1）确定SARS-COV-2包膜（E）蛋白的结构，该结构 组装成阳离子刺激宿主炎症体的阳离子选择通道； 2）调查 造成酸激活的四聚体质子的构成构成影响的构成构成影响的结构机制 影响Za病毒的渠道； 3）确定多药耐药细菌的结构 转运蛋白EMRE，以阐明质子耦合底物转运的机理。这些 膜蛋白 - E，M2和EMRE是遏制COVID-19大流行的药物靶标 在AIM 1中，我们将研究质子的结构基础 通过检查造成的B M2（BM2）突变体中的M2蛋白传导方向。野生型（WT）AM2 仅像转运蛋白一样向内进行质子，而WT BM2则在双向上进行质子，例如 规范渠道。这种差异与最近的数据相关，即AM2经历了交替访问 当BM2经历类似剪刀的运动以激活时，激活的运动。了解这些 差异，我们将研究一个BM2突变体，该突变体概括了AM2向内矫正表型。我们将 使用多维固态NMR光谱测量其结构和动力学 带有渠道活动的结构信息。在AIM 2中，我们将确定SARS-COV-2 E蛋白 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和具有结合抑制剂，以了解e的导致阳离子以及如何 电导可以阻塞。 2D和3D相关固态NMR实验将在 与通道活动测量的连接。在AIM 3中，我们将调查构象和 E通过31p和13c NMR的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，它正在介导病毒萌芽和释放。在AIM 4中， 我们将研究EMRE，它以质子耦合的方式排出阳离子药物以引起抗生素 大肠杆菌中的抗性。我们将采用多维19F NMR技术来测量蛋白质药物 限制底物结合口袋的结构的距离。这些研究应提供 详细的结构见解，对一些最具破坏性的膜运输机制 病毒和细菌，应建立药物设计的基础，以改善人类健康。