CryoEM analysis of Anthrax Toxin Pore Complexes

描述（由申请方提供）：本项目的主要目标是使用cryo-EM单颗粒分析获得插入脂质纳米盘的炭疽毒素保护性抗原（PA）孔复合物的更高分辨率结构（6- 10 E）。使用低温EM技术累积PA孔纳米盘图像数据集将允许我们获得PA-孔-纳米盘复合物的更高分辨率的结构细节（6-10 E）。正常模式柔性装配程序允许我们确定包含phe 427夹的柔性环的位置。我们将使用这种方法来具体定义重要的pH依赖性动态构象模式参与蛋白质易位，因为我们确定PA孔纳米盘结构的更高分辨率的结构。 具体地，我们将检查PA孔纳米盘复合物中发生的变化：1）当pH从7.0变化到5.5时，2）我们将比较野生型PA孔纳米盘结构与PA F427 A易位突变体纳米盘作为pH的函数。3）我们将确定结合至全长致死因子和N末端结构域的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结构元素涉及易位控制项目1和2。对于项目3，我们在构建PA孔纳米盘复合物方面的成功使我们具有独特的能力，可以研究当pH值从7降低到5.5时PA孔中可能的结构变化，并可能在PA孔-致死因子复合物中发生结构变化，模拟结合相互作用的变化和内体酸化期间发生的转变。研究和生物技术的影响：纳米盘炭疽毒素孔复合物可以作为这种毒素成分的活性形式的特异性抗原递送系统。 在固定条件下折叠膜蛋白防止聚集，但允许我们容易地将这些正确折叠的蛋白质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测定。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物（如梭菌毒素）的基础模型方法。纳米圆盘复合物将使我们能够探测和控制大群体或单个分子的结合相互作用的动力学和动力学。 这些特定的复合物可以作为开发高通量筛选平台以鉴定抗炭疽药物的平台。 公共卫生相关性：吸入炭疽杆菌孢子（一种潜在的生物武器）和随后产生的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观察炭疽毒素易位孔复合物（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炭疽治疗和新型疫苗。

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.