CryoEM analysis of Anthrax Toxin Pore Complexes

描述(申请人提供)：该项目的主要目标是使用冷冻EM单颗粒分析来获得插入到脂质纳米盘中的炭疽毒素保护性抗原(PA)孔复合体的更高分辨率结构(6-10e)。利用低温EM技术积累PA孔纳米盘图像数据集，将使我们能够获得PA-孔-纳米盘复合体(6-10E)的更高分辨率的结构细节。正常模式的柔性装配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孔纳米盘结构的较高分辨率结构时，我们将使用这种方法来具体定义蛋白质转运中涉及的重要的pH依赖的动态构象模式。具体地说，我们将研究PA孔纳米盘复合体中发生的变化1)随着pH从7.0变化到5.5，2)我们将比较野生型PA孔纳米盘结构与PA F427A易位突变纳米盘随pH的变化。3)我们将确定与全长致死因子和N末端结构域结合的PA孔纳米盘的络合物。这些比较的结构方法将使我们能够定义项目1和2中涉及易位控制的结构元素。对于项目3，我们成功构建PA孔纳米盘复合体使我们能够研究当pH从7降至5.5时PA孔中可能的结构变化，以及可能在PA孔-致死因子复合体中的结构变化，模拟结合相互作用和内体酸化过程中发生的转变的变化。研究和生物技术影响：纳米盘-炭疽毒素孔复合体可能作为这种毒素成分活性形式的特定抗原递送系统。在固定化条件下折叠的膜蛋白阻止了聚集，但允许我们很容易地将这些正确折叠的蛋白质插入到脂质纳米盘或其他脂质结构中，从而辅助膜蛋白结构的确定。我们的方法可以作为构建和研究其他细菌毒素大分子复合体(如梭状芽胞杆菌毒素)的基础模型方法。纳米盘复合体将使我们能够探测和控制大分子或单分子结合相互作用的动力学和动力学。这些特殊的复合体可以作为开发高通量筛选平台以识别抗炭疽预防药物的平台。 与公共卫生相关：吸入炭疽芽胞(一种潜在的生物武器)并随后产生外毒素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分离和查看炭疽毒素易位孔道复合体(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抗炭疽病药物和新的疫苗。

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.