CHARACTERIZATION OF THE TYPE IV PILUS MOTOR COMPLEX

IV 型皮鲁斯运动复合体的特征

• 批准号: RGPIN-2017-05757
• 负责人: Craig, Lisa
• 金额: $ 2.04万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711791
• 关键词: CHARACTERIZATION; TYPE; IV; PILUS; MOTOR

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.

IV型皮利是许多细菌表面上显示的细长细丝。它们具有多种功能，这些功能依赖于它们的以下能力：（i）粘附于各种基质，包括宿主细胞表面、来自附近细菌的皮利、DNA和噬菌体，以及（ii）去粘附或缩回，其沿着粘膜表面牵拉细菌沿着，将它们拉在一起形成保护性聚集体，并且可以将基质如DNA和噬菌体吸引到细菌中以获得营养和遗传变异。IV型皮利是数千个拷贝的主要菌毛蛋白亚基的聚合物。皮利通过一个复杂的分子机器在内膜上组装，该分子机器从细胞质到外膜跨越细菌包膜。我们的实验室是在这一领域的最前沿，与我们的IV型菌毛蛋白亚基的X射线晶体结构和完整的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理解细丝组装的框架。许多研究小组在了解菌毛组装装置的结构方面取得了显著的进展，但这些皮利组装的机制仍然是一个谜。这项建议的目的是表征菌毛机械内的分子马达复合物，以揭示它如何驱动细丝组装。 IV型皮利由菌毛蛋白亚基组装而成，所述菌毛蛋白亚基在菌毛组装之前通过其疏水性N-末端α-螺旋锚定在内膜中。聚合由内膜细胞质侧的六聚环状组装ATP酶提供动力。ATP水解诱导ATP酶的单个原聚体中的构象变化，其通过多位内膜平台蛋白中继到生长的菌毛，导致在每个亚基添加后细丝从膜的增量挤出。这些关键成分共同构成了运动复合体。我们推测，ATP酶原聚体中的连续ATP水解改变了它们对各个平台蛋白结构域的亲和力，导致它们围绕ATP酶环“行走”。平台蛋白围绕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，并在经过时将其向外挤出一小段距离。进入的菌毛蛋白亚基立即填充菌毛基部新打开的间隙，防止菌毛塌陷回膜中。我们将测试这个旋转菌毛组件模型的特征的电机复杂的组成部分，通过X射线晶体学，检查它们在体内和体外的相互作用，并测试旋转的电机组件使用光镊。我们将集中在霍乱弧菌毒素协同调节菌毛机制，因为它是最简单和最好的特点IV型菌毛系统之一。