NMR studies of bacterial needle and tip proteins

描述(申请人提供)：拟议的核磁共振研究的目标是确定在许多细菌病原体的发病机制中必不可少的细菌纳米注射器组装过程中涉及的蛋白质-蛋白质相互作用。纳米注射器是一个针状的大分子组件，构成了细菌蛋白质输出机制的结构部分，即III型分泌系统(T3SS)。T3SS还包括注入宿主细胞细胞质的效应蛋白，以及在细菌细胞质内与T3SS蛋白形成复合体的伴侣蛋白。T3SS针装置由20多种不同的蛋白质组装而成，由固定在细菌膜上的基本结构、位于细菌表面的针本身、尖端复合体和转位子组成。转位子是一种跨膜结构，它在宿主细胞膜上穿孔，允许细菌蛋白进入宿主细胞细胞质。在第一轮资助中，我们已经确定了针状蛋白与TIP蛋白的相互作用。目前，TIP蛋白如何与转位蛋白相互作用还完全不清楚。对于这一更新，我们的目标#1是确定TIP蛋白如何与转位蛋白相互作用。另一个未知的是TIP蛋白如何与它们的伴侣相互作用，因此我们的目标#2是确定TIP蛋白如何与它们的伴侣相互作用。为了促进我们对T3SS生物学的理解，我们的核磁共振研究将与电子显微镜和功能分析相补充。许多病原体依靠III型分泌系统感染全球数百万人。由于针具暴露在细菌表面，破坏针具组装是开发新型抗感染药物的一个有吸引力的目标。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。

DESCRIPTION (provided by applicant): The objective of the proposed NMR studies is to determine the protein-protein interactions involved in the assembly of a bacterial nanoinjector that is essential in the pathogenesis of many bacterial pathogens. The nanoinjector is a needle-like macromolecular assembly and forms the structural part of bacterial protein export machinery, the type III secretion system (T3SS). The T3SS also consists of effector proteins, which are injected into the host cell cytoplasm, and chaperones, which form complexes with T3SS proteins while inside the bacterial cytoplasm. The T3SS needle apparatus is assembled from over 20 different proteins and consists of a basal structure anchored on the bacterial membranes, the needle itself on the bacterial surface, a tip complex, and a translocon. The translocon is the membrane spanning structure that punctures a hole on the host cell membrane to allow the passage of bacterial proteins into the host cell cytoplasm. On the first round of funding, we have determined how the needle protein interacts with the tip protein. Currently, how the tip protein interacts with the translocon is completely unknown. For this renewal, our Aim #1 is to determine how the tip protein interacts with the translocon proteins. Another unknown is how the tip proteins interact with their chaperones, thus our Aim #2 is to determine how the tip proteins interact with their chaperones. To advance our understanding of the biology of the T3SS, our NMR studies will be complemented with electron microscopy and functional assays. Many pathogens rely on the type III secretion system to infect millions of people worldwide. Because the needle apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive target for the development of novel anti- infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly.