CHARACTERIZATION OF THE TYPE IV PILUS MOTOR COMPLEX

IV型菌毛是长而细的细丝，显示在许多细菌的表面。它们具有多种功能，这些功能依赖于它们(i)粘附各种底物的能力，包括宿主细胞表面、附近细菌的菌毛、DNA和噬菌体，以及（ii）解聚合或缩回，这将细菌沿着粘膜表面拉紧，将它们拉紧在一起形成保护性聚集体，并将DNA和噬菌体等底物吸引到细菌中进行营养和遗传变异。IV型菌毛是由数千个主要菌毛亚基的拷贝组成的聚合物。菌毛是由一个复杂的分子机器从细胞质跨越细菌包膜到外膜组装在细胞膜上的。我们的实验室处于这一领域的前沿，我们的IV型菌毛蛋白亚基的x射线晶体结构和完整菌毛细丝的低温电子显微镜重建提供了这些细胞器的原子分辨率图片和理解细丝组装的框架。许多研究小组在了解毛组装装置的结构方面取得了显著进展，但这些毛组装的机制仍然是一个谜。本提案旨在表征毛丝机械中的分子马达复合物，以揭示它如何驱动灯丝组装。*** IV型菌毛由菌毛亚基组装而成，在菌毛组装之前，它们通过疏水的n端α -螺旋锚定在内膜上。聚合是由细胞膜细胞质侧的六聚体环状组装atp酶驱动的。ATP水解诱导ATP酶的单个原形成物的构象变化，这种变化通过多面体的内膜平台蛋白传递给生长的菌毛，导致每增加一个亚基，就会从膜中挤出长丝。这些关键成分共同构成了运动复合体。我们假设ATP酶原体中的连续ATP水解改变了它们对单个平台蛋白结构域的亲和力，导致它们在ATP酶环周围“行走”。平台蛋白在生长的菌毛底部的圆周运动使其小的质周环与菌毛底部接触，在经过时将其向外挤压一小段距离。一个进入的菌毛亚基立即填补菌毛底部新打开的空隙，防止菌毛塌陷回膜中。我们将通过x射线晶体学表征运动复合体的组成部分，检查它们在体内和体外的相互作用，并使用光学镊子测试运动组件的旋转，来测试这种旋转毛毛组装模型。我们将重点关注霍乱弧菌毒素协同调节的菌毛机制，因为它是最简单和最具特征的IV型菌毛系统之一。********

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.********