CryoEM analysis of Anthrax Toxin Pore Complexes

炭疽毒素孔隙复合物的冷冻电镜分析

• 批准号: 8108210
• 负责人: Mark T Fisher
• 金额: $ 22万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8108210
• 关键词: Anthrax disease; Antigens; Arizona; Bacillus anthracis; Bacillus anthracis spore; Bacteremia; Bacterial Toxins; Binding; Biological Assay; Biotechnology; Bioterrorism; Breathing; Cessation of life; Chemicals; Clostridium; Collaborations; Complex; Computer Simulation; Coupled; Cryoelectron Microscopy; Cytoplasm; Data Set; Detergents; Development; Disease; Elements; Endosomes; Environment; Exotoxins; Foundations; Goals; Image; Infection; Kinetics; Knowledge; Length; Lipid Bilayers; Lipids; Livestock; Location; Macromolecular Complexes; Medical; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Movement; N-terminal; Negative Staining; Organism; Population; Pore Proteins; Procedures; Production; Property; Protein translocation; Proteins; Publishing; Reaction; Reproduction spores; Research; Research Personnel; Resolution; Screening procedure; Septic Toxemia; Structure; Surface; System; Techniques; Testing; Time; Toxin; Universities; Work; anthrax lethal factor; anthrax toxin; antigen binding; base; comparative; edema factor; flexibility; high throughput screening; inhibitor/antagonist; mutant; nanodisk; novel; novel therapeutics; novel vaccines; particle; prevent; prophylactic; protein folding; protein structure; scaffold; single molecule; small molecule; success; three dimensional structure

DESCRIPTION (provided by applicant): The primary goal of this project is to use cryo-EM single particle analysis to obtain higher resolution structures (6-10E) of the anthrax toxin protective antigen (PA) pore complex inserted into lipid nanodiscs. Accumulating PA pore nanodisc image data sets using cryo EM techniques will allow us to obtain higher resolution structural detail of the PA-pore -nanodisc complex (6-10 E). Normal mode flexible fitting procedures have allowed us to determine the location of the flexible loop containing the phe427 clamp. We will use this method to specifically define the important pH dependent dynamic conformational modes involved in protein translocation as we determine the higher resolution structures of the PA pore nanodisc structure. Specifically, we shall examine the changes that occur in the PA pore nanodisc complexes 1) as pH changes from 7.0 to 5.5, 2) We shall compare the wild type PA pore nanodisc structures with the PA F427A translocation mutant nanodisc as a function of pH. 3) We shall determine the complexes of the PA pore nanodisc bound to the full length lethal factor and the N terminal domain. These comparative structural approaches will enable us to define structural elements involved in translocation control for projects 1 and 2. For project 3 our success at constructing PA pore nanodisc complexes gives us the unique ability to investigate a possible structural change in PA pore and possibly in PA pore-lethal factor complexes when the pH is decreased from 7 to 5.5, mimicking changes in binding interactions and transitions that occur during endosome acidification. Research and Biotechnology implications: The nanodisc-anthrax toxin pore complex may serve as a specific antigen delivery system for the active form of this toxin component. Folding membrane proteins under immobilizing conditions prevents aggregation but allows us to easily insert these properly folded proteins into lipid nanodiscs or other lipid structures thus aiding membrane protein structure determination. Our methods may serve as a basis model method for constructing and studying other bacterial toxin macromolecular complexes (e.g. Clostridia Toxin). Nanodisc complexes will enable us to probe and control kinetic and dynamics of binding interactions for large populations or single molecules. These particular complexes could serve as platforms for the development of high throughput screening platforms to identify anti-anthrax prophylactics. PUBLIC HEALTH RELEVANCE: Inhalation of the Bacillus anthracis spores, a potential bioweapon and subsequent production of exotoxin causes severe toxemia, bacteremia and death. We are implementing novel methods to isolate and view the structure of the anthrax toxin translocation pore complex (Protective Antigen) to apply this new structural knowledge toward developing new therapeutic anti-anthrax treatments and novel vaccines.

描述（由申请人提供）：该项目的主要目标是使用冷冻EM单个粒子分析获得炭疽毒素保护抗原（PA）孔复合物的更高分辨率结构（6-10E），这些孔隙插入了脂质纳米盘中。使用冷冻技术累积PA孔纳米轴图像数据集将使我们能够获得PA孔 - 纳米盘复合物的更高分辨率结构细节（6-10 E）。正常模式柔性拟合程序使我们能够确定包含PHE427夹具的柔性环的位置。当我们确定PA孔纳米轴结构的较高分辨率结构时，我们将使用此方法专门定义参与蛋白质易位的重要pH依赖性动态构象模式。 具体而言，我们将检查PA孔纳米盘复合物中发生的变化1）pH从7.0变为5.5，2）我们应将野生型PA PA孔纳米散发结构与PA F427A易位nanodant cyptake进行比较。 3）我们将确定与全长致死因子和N末端结构域结合的PA孔纳米盘的复合物。这些比较结构的方法将使我们能够定义项目1和2的易位控制的结构要素。对于项目3，我们成功地构建PA孔纳米散发液轴复合物使我们具有独特的能力，可以研究PA孔中可能的结构变化，可能在PA孔孔致死因子复合物中降低7至5.5的pH值在7到5.5，模拟构成互动互动互动互动和构成构成之间的变化。研究和生物技术的意义：纳米轴 - 蒽毒素孔复合物可以作为该毒素成分的主动形式的特定抗原递送系统。 在固定条件下折叠膜蛋白可以防止聚集，但使我们能够轻松地将这些正确折叠的蛋白插入脂质纳米盘或其他脂质结构中，从而有助于膜蛋白结构的确定。我们的方法可以用作构建和研究其他细菌毒素大分子复合物（例如梭状芽胞杆菌毒素）的基础模型方法。纳米盘复合物将使我们能够为大种群或单个分子探测和控制结合相互作用的动力学和动力学。 这些特殊的配合物可以用作开发高吞吐量筛查平台以识别预防抗炎药的平台。 公共卫生相关性：吸入炭疽芽孢杆菌孢子，潜在的生物武器以及随后的外毒素的产生会导致严重的毒血症，菌血症和死亡。我们正在实施新的方法来隔离和查看炭疽毒素易位孔隙复合物（保护性抗原）的结构，以将这种新的结构知识应用于开发新的治疗性抗疫次治疗和新型疫苗。