Catalytic mechanism of the usher in pilus biogenesis by uropathogenic E. coli

 描述（由申请人提供）：尿路感染（UTI）是世界上最常见的细菌感染之一，导致超过15亿美元的相关医疗费用。尿路感染最常见的原因是尿路致病性大肠杆菌（UPEC）。UPEC通过分子伴侣/引导（CU）途径组装并分泌皮利到其细胞表面。这些皮利是粘附相关的结构，以毛发状的方式从细胞表面突出并介导对尿路的粘附。CU途径的伴侣蛋白和引导蛋白协同工作以构建这些皮利。分子伴侣，周质蛋白，促进亚基折叠，防止过早的亚基-亚基相互作用，并将亚基靶向外膜引导蛋白。Usher是一个多功能的菌毛组装和分泌平台，催化分子伴侣-亚基交换为亚基-亚基相互作用以组装菌毛纤维，并为纤维分泌到细胞表面提供通道。usher是一种大蛋白，由周质N-末端结构域（N）、由内部Plug结构域门控的跨膜β-桶结构域和两个周质C-末端结构域（C1和C2）组成。这些结构域共同作用以促进菌毛的有序组装和分泌。这个建议的总体目标是从结构和机制上描述引座员在菌毛生物发生中的作用。该提议将测试以下假设：引座员既是门控分泌通道又是催化纳米机器，并且其催化活性是由于引座员-分子伴侣-亚基相互作用的仔细协调序列以及分子伴侣-亚基复合物相对于彼此的准确放置。我建议使用荧光技术来了解如何引导转移菌毛亚基从其N域的C域，一个必要的步骤，以清除N域的下一个菌毛亚基的掺入。我将使用类似的技术来理解Plug与未激活的usher的C域的相互作用，以及Plug域如何以及何时在usher激活时从usher通道中移除。最后，我建议探索usher催化菌毛组装和分泌的结构基础。我将使用分子技术来产生适合于冷冻电子显微镜和X射线晶体学分析的菌毛组装中间体。该提案中描述的实验所获得的信息将导致蛋白质分泌领域的新进展。这些结果将有助于确定针对UPEC的新型治疗方法的物理和机制靶点，这在当前抗生素耐药性猖獗的时代是一项特别重要的奋进。

 DESCRIPTION (provided by applicant): Urinary tract infections (UTIs) are among the most common bacterial infections in the world and result in more than $1.5 billion USD in associated medical costs. The most common cause of UTIs is uropathogenic Escherichia coli (UPEC). UPEC use the chaperone/usher (CU) pathway to assemble and secrete pili onto their cell surfaces. These pili are virulence-associated structures that project away from the cell surface in a hairlike fashion and mediate adhesion to the urinary tract. The chaperone and usher proteins of the CU pathway work in unison to construct these pili. The chaperone, a periplasmic protein, facilitates subunit folding, prevents premature subunit-subunit interactions, and targets subunits to the outer membrane usher protein. The usher is a multifunctional pilus assembly and secretion platform, catalyzing the exchange of chaperone-subunit for subunit-subunit interactions to assemble the pilus fiber and providing the channel for secretion of the fiber to th cell surface. The usher is a large protein consisting of a periplasmic N-terminal domain (N), a transmembrane beta-barrel domain that is gated by an internal Plug domain, and two periplasmic C-terminal domains (C1 and C2). These domains act together to facilitate the ordered assembly and secretion of the pilus. The overall goal of this proposal is to structurally and mechanistically characterize the usher's role in pilus biogenesis. This proposal will test the hypothesis that the usher is both a gated secretion channel and catalytic nanomachine, and that its catalytic activity is due to a carefully coordinated sequence of usher-chaperone-subunit interactions and the accurate placement of chaperone-subunit complexes relative to one another. I propose to use fluorescence techniques to understand how the usher transfers pilus subunits from its N domain to its C domains, a necessary step to clear the N domain for incorporation of the next pilus subunit. I will use similar techniques to understand the Plug's interaction with the C domains of the inactivated usher and how and when the Plug domain is removed from the usher channel upon usher activation. Finally, I propose to probe the structural basis for usher-catalyzed pilus assembly and secretion. I will use molecular techniques to generate pilus assembly intermediates suitable for analysis by cryo-electron microscopy and X-ray crystallography. The information gained by the experiments described in this proposal will lead to new advances in the field of protein secretion. The results will aid in the identification f physical and mechanistic targets for novel therapeutics against UPEC, a particularly important endeavor in the current era of rampant antibiotic resistance.