CryoEM analysis of Anthrax Toxin Pore Complexes

描述（由申请人提供）：该项目的主要目标是使用冷冻电镜单粒子分析来获得插入脂质纳米圆盘中的炭疽毒素保护性抗原（PA）孔复合物的更高分辨率结构（6-10E）。使用冷冻电镜技术积累 PA 孔纳米盘图像数据集将使我们能够获得 PA-孔-纳米盘复合物 (6-10 E) 的更高分辨率的结构细节。正常模式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 孔纳米盘结构的更高分辨率结构时，我们将使用这种方法来具体定义参与蛋白质易位的重要 pH 依赖性动态构象模式。 具体来说，我们将检查 PA 孔纳米圆盘复合物 1) 当 pH 从 7.0 变化到 5.5 时发生的变化，2) 我们将比较野生型 PA 孔纳米圆盘结构与 PA F427A 易位突变体纳米圆盘作为 pH 的函数。 3) 我们将确定与全长致死因子和 N 末端域结合的 PA 孔纳米圆盘的复合物。这些比较结构方法将使我们能够定义涉及项目 1 和 2 的易位控制的结构元件。对于项目 3，我们成功构建了 PA 孔纳米圆盘复合物，这使我们具有独特的能力来研究当 pH 从 7 降至 5.5 时 PA 孔以及 PA 孔致死因子复合物中可能的结构变化，模拟内体酸化过程中发生的结合相互作用和转变的变化。研究和生物技术意义：纳米盘-炭疽毒素孔复合物可以作为该毒素成分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。我们的方法可以作为构建和研究其他细菌毒素大分子复合物（例如梭菌毒素）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量群体或单个分子的结合相互作用的动力学和动力学。 这些特殊的复合物可以作为开发高通量筛选平台的平台，以鉴定抗炭疽预防剂。 公共卫生相关性：吸入炭疽杆菌孢子（一种潜在的生物武器）以及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，以应用这种新的结构知识来开发新的抗炭疽治疗方法和新型疫苗。

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.