细菌六型分泌系统基板复合体TssF-G-K的结构解析及TssK与系统其它核心蛋白相互作用的研究

Type VI secretion system (T6SS), a recently identified Gram-negative bacterial secretion system, is widely distributed in diverse bacteria species. Previous studies revealed that T6SS is a complex machine consisting of a phage like injection complex injecting the effectors into target cells and a membrane complex which anchoring the needle complex to the membrane. Though the membrane complex consists of TssJ, TssL and TssM, and the phage like injection complex includes a sheath assembled by TssB-C and a needle structure assembled by Hcp (TssD) and VgrG (TssI) are studied intensively, the components and assembly of baseplate of T6SS remains largely unclear. Studies have showed that baseplate protein TssK interacts with both the membrane complex and the needle complex, and also could form a stable complex with TssF and TssG. Since no structural information is available for TssK, the interaction mode of TssK with these interacted proteins or complex remains largely unclear. Fortunately, we just solved the crystal structure of the TssK and found the differences from the reported hypothesis on the baseplate formation. In this project, to dissect the interaction between TssK and TssA, TssB-C, Hcp，TssE and TssL, different length constructs of TssK including different domains and different point mutation mutants will be designed based on our recently solved crystal structure of TssK; and the interaction between TssK constructs and these proteins will be analyzed. At the same time, x-ray crystallography method will be used to solve the crystal structure of TssF, and x-ray crystallography and Cryo-EM method will be also applied to determine the TssF-G subcomplex and TssF-G-K complex structure. Our results will elucidate the interaction between TssK and TssA, TssB-C, Hcp,TssE, TssL and TssF-G, and provide insight into the assembly of the baseplate of T6SS, and help to understand the assembly of the whole complex of T6SS and the evolution of T6SS.

六型分泌系统(T6SS)是存在于革兰氏阴性菌中的分泌系统。该系统是由一个类似噬菌体尾部的复合体和一个跨膜复合体组成的复杂的分子机器。目前，T6SS跨膜复合体及针管的结构组成较为清楚，另外基板蛋白TssK能够与TssJ-L-M，TssB-C和Hcp-VgrG-PAAR复合体相互作用，并与TssF和TssG形成稳定的复合体，但基板的具体组成及各组分的结构仍不清楚。我们刚刚解析了TssK的晶体结构，发现TssK与已报导的T4噬菌体的基板蛋白没有同源性，因此本项目拟在TssK结构的基础上，结合晶体学方法及冷冻电镜法解析TssF，TssF-G及TssF-G-K的结构，并根据TssK结构域及可能与其它蛋白相互作用的位点构建含不同结构域的蛋白，并通过测定它们与其他蛋白相互作用，从而明确它们之间的相互作用位点和关系，明确TssK与其它蛋白的作用方式并阐明T6SS的基板的组成,揭示其与噬菌体基板结构的异同。

本项目主要针对六型分泌系统的结构与功能以及调控方式进行深入研究。TssK对于T6SS的分泌是必须的，本项目解析了首个全长的TssK的晶体结构，通过对其结构的分析，发现K367的突变会导致T6SS的功能丧失。通过基因缺失突变和基因回补的方法，明确了六型分泌系统的PpkA对六型分泌系统的调控具有非常重要的功能。解析了长度为294个氨基酸残基的PpkA的激酶结构域的结构，同时解析了其结合不同的ATP类似物的晶体结构，明确了该激酶处于自抑制状态，对该结构进行分析以及和其他结构进行比对揭示了该激酶的抑制机制是一种普遍的抑制机制；RtkS能够稳定PpkA，为了明确其稳定PpkA的机制，我们解析了RtkS的晶体结构和RtkS与PpkA的C端结构域的复合体的晶体结构。TagF抑制T6SS的分泌，运用pull-down方法，我们发现TagF与TssB/TssC，HCP等相互作用，细菌双杂表明TagF与HCP的相互作用最强，进一步的研究表明TagF通过与HCP的相互作用抑制T6SS的分泌，并且发现HCP的C末端是与TagF相互作用所必须的。基因缺失和回补研究发现双组份调控系统CpxR/A及BarA/UvrY正调控T6SS的分泌，进一步的研究发现CpxR/A在转录水平上调控T6SS，CpxA的激酶活性对于调控并不重要，而CpxR的磷酸化则对T6SS的转录调控是必须的，CpxA的磷酸酶活性是CpxA调控T6SS所必须的。BarA/UvrY调控T6SS的hcp和两个vgrG基因的转录，BarA的激酶活性和UvrY的磷酸化位点对于调控都是必须的，UvrY通过直接结合这些基因的启动子来调控它们的转录。通过构建不同的vgrG基因的缺失突变株，和回补实验，我们发现T6SS的两个VgrG协同完成T6SS的分泌，对未知功能基因的鉴定发现13845基因通过与VgrG协同影响T6SS的分泌。为了深入研究13845的功能，还解析了13845的晶体结构。本项目的研究结果完善和加深了我们对T6SS系统的结构及其调控机制的认识。