NMR studies of bacterial needle and tip proteins

描述（由申请人提供）：拟议研究的目的是了解细菌III型分泌针装置组装中涉及的蛋白质-蛋白质相互作用。这种装置类似于细菌表面的注射器，被许多病原体用来将毒力因子注入靶细胞以引发人类疾病。这种针状的组装由大约120个相同的蛋白质组成，它们以超螺旋的方式排列，针尖上覆盖着尖端蛋白质。在过去的两年里，我们已经完成了两个针状单体的核磁共振结构：来自伪伯克霍尔德菌的BsaL，一种与生物战相关的病原体，以及来自鼠伤寒沙门氏菌的PrgI，一种与食物中毒相关的病原体。我们还利用核磁共振鉴定了参与福氏志贺氏菌针尖蛋白结合相互作用的关键残基。我们的目标是通过确定涉及针和尖端蛋白质的精确蛋白质接触来阐明针装置是如何组装的。我们将结合核磁共振与电子显微镜和分子模型来确定组装针的结构。我们的目标是：(1)确定针头是如何组装的，(2)确定尖端蛋白质是如何在针头上对接的，(3)使用诱变和微生物分析将结构结果与针头组装和体内毒力联系起来。许多病原体依靠针头装置感染全世界数百万人，其中许多是生物恐怖主义的潜在媒介。由于这种装置暴露在细菌表面，破坏针头组装是开发新型抗感染药物的一个有吸引力的方法。这种方法需要详细了解针组装中涉及的蛋白质-蛋白质相互作用。许多耐药细菌病原体的出现给公共卫生和安全带来了重大问题，其中许多细菌依靠针状蛋白质组装来感染宿主。拟议的研究旨在通过确定其组成部分的蛋白质-蛋白质相互作用来阐明这种针是如何组装的。这些知识将用于设计新的抗菌剂。

DESCRIPTION (provided by applicant): The objective of the proposed studies is to understand the protein-protein interactions involved in the assembly of the bacterial type III secretion needle apparatus. This apparatus resembles a syringe on the bacterial surface and is used by many pathogens to inject virulence factors into target cells to initiate human diseases. The needle-like assembly consists of about 120 copies of identical proteins that are arranged in a superhelical manner, and the apex of this needle is capped by tip proteins. Over the past two years, we have completed the NMR structures of two needle monomers: BsaL from Burkholderia pseudomallei, a pathogen associated with biowarfare, and PrgI from Salmonella typhimurium, a pathogen associated with food poisoning. We have also used NMR to identify the key residues involved in the binding interaction between the needle and tip proteins of Shigella flexneri. Our goal is to elucidate how the needle apparatus is assembled by determining the precise protein-protein contacts involving the needle and tip proteins. We will combine NMR with electron microscopy and molecular modeling to determine the structure of the assembled needle. Our aims are: (#1) determine how the needle is assembled, (#2) determine how the tip protein docks on the needle, and (#3) use mutagenesis and microbiological assays to correlate the structural results to needle assembly and virulence in vivo. Many pathogens rely on the needle apparatus to infect millions of people worldwide and many are potential agents of bioterrorism. Because this apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive approach for the development of novel anti-infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly. PUBLIC HEALTH RELEVANCE The appearance of many antibiotic resistant bacterial pathogens poses a major problem in public health and safety and many of these bacteria rely on a needle-like protein assembly to infect their hosts. The proposed research seeks to elucidate how this needle is assembled by determining the protein-protein interactions of its components. This knowledge will then be used in the design of novel anti-bacterial agents.