病原菌中PPX-GppA蛋白酶的结构与作用机理

Inorganic polyphosphate (poly-P) is a simple molecule with a complex range of biological functions. It is fundamentally important to all cellular life forms. In bacteria, poly-P helps cells to survive in hostile conditions, e.g. during desiccation, nutritional starvation, or when being ‘attacked’ with antibiotic agents. Furthermore, poly-P also plays a pivotal role in the ability of pathogenic (disease-causing) bacteria to cause persistent or ‘virulent’ infections via biofilm formation. However, the precise molecular mechanisms underlying the role of poly-P remain obscure. Polyphosphate hydrolase proteins (PPX-GppA family) are responsible for the hydrolysis of poly-P molecules in most bacteria. The general consensus is that bacterial PPX-GppA proteins also have the universal ability to hydrolyze guanosine pentaphosphate (pppGpp) intracellular signaling molecules, which are also known as ‘magic spot’ alarmones. These molecules play various key roles in bacterial survival and adaptation. The general paradigm is that pppGpp molecules inhibit the poly-P degrading activities of all PPX-GppA proteins. Thus PPX-GppA proteins are posited to act as ‘gatekeepers’ between two vitally important cellular pathways. However, our preliminary results show that this general paradigm is not universally true, and must be critically re-evaluated. In this proposal, we will establish structure-function relationships to clarify the biological roles of bacterial PPX-GppA proteins across bacteria. This will be done using a focused combination of biochemical, genetic, bioinformatics and structural biology approaches. We will characterize the in vitro biochemical activity of representative PPX-GppA enzymes from Mycobacterium smegmatis, Pseudomonas aeruginosa, Vibrio cholera and Porphyromonas gingivalis; determine their crystal structures alone or in complex with bound substrates; examine their roles in response to various bacterial stresses in vivo; and combine the structural and function data to validate PPX-GppA enzymes as potential drug targets. This project is an example of structural biology contributing to microbial physiology studies and should provide a more complete understanding of the role of PPX-GppA enzymes in bacterial survival and biofilm formation.

多聚磷酸（poly-P）在细菌中具有重要的生理功能。Poly-P的水解主要由聚磷酸水解酶蛋白家族（PPX-GppA）完成。PPX-GppA对病原菌的生物膜形成能力、致病性、耐药性等方面起到重要作用。迄今对病原菌PPX-GppA蛋白的研究还非常有限，缺乏结构与功能的相关性研究。本项目以铜绿假单胞菌、霍乱弧菌、牙龈卟啉单胞菌和结核分枝杆菌4种典型病原菌的PPX-GppA蛋白为研究对象，综合利用微生物学、结构生物学、分子生物学等多种研究方法，解析不同病原菌PPX-GppA蛋白结构，阐明结构与功能的关系，剖析poly-P和(p)ppGpp之间的相互作用，揭示PPX-GppA调节细菌poly-P水平、生长速率、压力耐受性以及生物膜形成的机理，确定潜在的药物靶点，筛选有效抑制物。本项目将结构生物学研究手段应用到微生物生理生化研究，充分体现结构与功能研究相结合的优势，为解决更复杂的微生物学问题奠定基础。

病原菌严重威胁人类健康，其应对外界压力、生物膜的形成、致病性和抗药性都受到多聚磷酸以及多聚磷酸外切酶PPX-GppA的调控。目前关于PPX-GppA蛋白的结构和功能研究还很少。本项目围绕病原菌的PPX-GppA蛋白的结构和功能开展研究。项目解析了耻垢分枝杆菌中MS-PPX蛋白及其与小分子抑制物的复合物结构，分析了其活性中心和底物结合位点。解析了MS-PPX的突变体R273A的三维晶体结构，通过与天然状态结构的对比，分析其可能的结构构象变化方式。根据结构和功能数据，确定了MS-PPX关键氨基酸残基的重要作用，探讨了其催化作用机制。解析了牙龈卟啉单胞菌中PgPPX的三维晶体结构，解析了Pg-PPX的2种突变体E118A和R255A的三维结构，以及 E118A与底物短链聚磷酸Poly-P3的复合物结构，根据结构和功能数据，对比不同结构的变化，提出了PgPPX可能的催化作用机制。解析了霍乱弧菌、铜绿假单胞菌和运动发酵单胞菌3种“长链”PPX-GppA蛋白的三维晶体结构，分析了可能的活性中心和关键位点。本项目通过解析5种细菌PPX-GppA蛋白及其与底物的复合物三维结构，从结构生物学角度解释PPX-GppA如何实现对多聚磷酸的水解，从而建立PPX-GppA蛋白的催化分子机制，分析了不同长度 PPX-GppA蛋白的区别，确定了源自不同病原菌的PPX-GppA蛋白的关键催化结构域以及活性位点，阐明了它们的催化活性和生理学功能，重点分析了PPX-GppA影响病原菌适应性的分子机制，并进一步将结构与功能结合，筛选潜在的药物靶点，进行以结构为基础的有效抑制物设计，为新型药物研发奠定基础。此外鉴于poly-P 在微生物细胞中的广泛存在和重要作用，本研究结果也将为包括废水的脱磷、生物修复、磷元素循环等微生物学等其他领域的研究提供有效的数据支持。