CHARACTERIZATION OF THE TYPE IV PILUS MOTOR COMPLEX

IV型菌毛是许多细菌表面显示的细长细丝。它们具有一系列不同的功能，这依赖于它们的能力：(I)附着在各种底物上，包括宿主细胞表面、附近细菌的菌毛、DNA和噬菌体，以及(Ii)解聚或收回，这将细菌沿着粘膜表面拉近，将它们拉近在保护性聚集体中，并能将DNA和噬菌体等底物吸引到细菌中进行营养和遗传变异。IV型菌毛是主要菌毛蛋白亚单位的数千个拷贝的聚合物。菌毛由一个复杂的分子机器组装在内膜上，分子机器从细胞质跨越细菌包膜到外膜。我们的实验室处于这一领域的前沿，我们的IV型菌毛蛋白亚单位的X射线晶体结构和完整菌毛细丝的冷冻电子显微镜重建提供了这些细胞器的原子分辨率图片和了解细丝组装的框架。许多研究小组在了解菌毛组装装置的结构方面已经取得了显著的进展，但这些菌毛的组装机制仍然是一个谜。这项建议旨在表征菌毛机械中的分子马达复合体，以揭示它是如何驱动细丝组装的。*IV型菌毛由菌毛蛋白亚单位组装而成，在菌毛组装之前，菌毛蛋白亚单位通过疏水的N-末端α-螺旋锚定在内膜上。聚合是由内膜细胞质一侧的六聚体环状组装ATPase提供动力的。ATP水解会诱导ATPase的各个原型的构象变化，这些构象通过一个多位的内膜平台蛋白传递到生长中的菌毛，导致在每个亚基添加时从膜上逐渐挤出细丝。这些关键部件共同构成了发动机复合体。我们假设，在ATPase原分子中顺序的ATP水解改变了它们对单个平台蛋白结构域的亲和力，导致它们“走”在ATPase环周围。平台蛋白围绕正在生长的菌毛底部的这种圆周运动使其小的周质环与菌毛底部接触，在菌毛经过时将其向外挤出一小段距离。一个输入的菌毛素亚单位会立即填补菌毛底部新打开的缝隙，防止菌毛塌陷回到膜中。我们将通过X射线结晶学表征马达复合体的组件，检查它们在体内和体外的相互作用，并使用光学镊子测试马达组件的旋转，来测试这种旋转毛发组装模型。我们将重点介绍霍乱弧菌毒素共调控菌毛机制，因为它是IV型菌毛系统中最简单和最具特点的之一。

Type IV pili are long thin filaments displayed on the surfaces of many bacteria. They have a diverse array of functions that rely on their ability to (i) adhere to various substrates, including host cell surfaces, pili from nearby bacteria, DNA and bacteriophage, and (ii) to depolymerize or retract, which pulls the bacteria along mucosal surfaces, pulls them close together in protective aggregates, and can draw substrates like DNA and phage into the bacterium for nutrition and genetic variation. Type IV pili are polymers of thousands of copies of the major pilin subunit. The pili are assembled at the inner membrane by a complex molecular machine spanning the bacterial envelope from the cytoplasm to the outer membrane. Our lab is at the forefront of this field, with our x-ray crystal structures of Type IV pilin subunits and cryo-electron microscopy reconstructions of intact pilus filaments providing an atomic resolution picture of these organelles and a framework for understanding filament assembly. Remarkable progress has been made by many groups in understanding the architecture of the pilus assembly apparatus, yet the mechanism by which these pili are assembled remains a mystery. This proposal aims to characterize the molecular motor complex within the pilus machinery to reveal how it drives filament assembly.*** Type IV pili are assembled from pilin subunits, which are anchored in the inner membrane via their hydrophobic N-terminal alpha-helices prior to pilus assembly. Polymerization is powered by a hexameric ring-shaped assembly ATPase on the cytoplasmic side of the inner membrane. ATP hydrolysis induces a conformational change in individual protomers of the ATPase, which is relayed to the growing pilus through a polytopic inner membrane platform protein, resulting in incremental extrusion of the filament from the membrane upon each subunit addition. These critical components together form the motor complex. We hypothesize that sequential ATP hydrolysis in the ATPase protomers alters their affinity for the individual platform protein domains, causing them to “walk” around the ATPase ring. This circular motion of the platform protein around the base of the growing pilus brings its small periplasmic loop into contact with the bottom of the pilus, extruding it outward a short distance as it passes. An incoming pilin subunit immediately fills the newly opened gap at the base of the pilus, preventing the pilus from collapsing back into the membrane. We will test this rotary pilus assembly model by characterizing the components of the motor complex by x-ray crystallography, examining their interactions both in vivo and in vitro, and testing for rotation of the motor components using optical tweezers. We will focus on the Vibrio cholerae toxin-coregulated pilus machinery as it is one of the simplest and best characterized of the Type IV pilus systems.********