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型分泌物针具组装过程中涉及的蛋白质-蛋白质相互作用。这种装置就像细菌表面的注射器，被许多病原体用来向目标细胞注入毒力因子，从而引发人类疾病。这个针状的组装由大约120个相同的蛋白质以超螺旋方式排列组成，这个针尖被尖端的蛋白质覆盖。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：来自与生物武器相关的伯克霍尔德氏菌的Bsal和来自与食物中毒有关的鼠伤寒沙门氏菌的PrgI。我们还利用核磁共振技术确定了参与志贺氏菌针尖蛋白相互作用的关键残基。我们的目标是通过确定涉及针头和针尖蛋白质的精确蛋白质接触来阐明针头装置是如何组装的。我们将结合核磁共振、电子显微镜和分子建模来确定组装的针的结构。我们的目标是：(#1)确定针是如何组装的，(#2)确定末端蛋白如何对接在针上，以及(#3)使用诱变和微生物学试验将结构结果与针组装和体内毒力相关联。许多病原体依靠针头设备感染全球数百万人，其中许多是生物恐怖主义的潜在病原体。由于这种装置暴露在细菌表面，扰乱针头组装是开发新型抗感染药物的一个有吸引力的方法。这种方法需要对针头组装中涉及的蛋白质-蛋白质相互作用有详细的了解。公共卫生相关性许多抗药性细菌病原体的出现对公共健康和安全构成了一个重大问题，其中许多细菌依赖针状蛋白质组装来感染宿主。这项拟议的研究试图通过确定其组件的蛋白质-蛋白质相互作用来阐明这种针是如何组装的。然后，这些知识将被用于设计新型抗菌剂。

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.