结核分枝杆菌毒素蛋白MazF-mt识别底物RNA的结构生物学机制

Toxin-antitoxin (TA) system MazF-MazE plays key roles in bacterial physiological processes, such as programmed cell death, stress responses and persistence. MazF can function as a toxin to cleavage their own or foreign RNA by its RNA endonuclease activity for translation inhibition, while the antitoxin MazE can inhibit MazF activity via their direct interactions. The Mycobacterium tuberculosis genome harbors nine TA systems that are members of the mazEF family (MazF-mt1 through MazF-mt9), whereas most prokaryotes only have one or two. The toxins MazF-mt3, MazF-mt6 and MazF-mt9 can promote M. tuberculosis drug tolerance and virulence in guinea pigs. Each MazF-mt toxin appears to recognize and cleave a unique RNA sequence and their substrate cover mRNA, rRNA and tRNA. In this project, we aim to obtain the crystal structures and solution conformations of MazF-mt3, MazF-mt6 and MazF-mt9 in complex with their respective substrate RNA or ribosome. These structures will be solved by protein crystallography and small-angle X-ray scattering (SAXS) in the beamline stations of Shanghai Synchrotron Research Facility (SSRF) and Beijing Synchrotron Radiation Facility (BSRF). On this basis, we are going to perform the in vivo and in vitro interaction studies between the toxins and their substrate RNA by site-directed mutagenesis and the resulting effects on host physiological processes. The results will reveal the molcular mechanisms of various substrate RNA recognizition by these toxins. Our findings will suggest new strategies for the discovery of potential antibiotics targeting this TA system, and may provide novel insights into clinical drug resistance management of M. tuberculosis.

毒素-抗毒素系统MazEF与细菌的细胞程序性死亡、应激反应和耐药性等生理功能密切相关。毒素蛋白MazF通过其RNA核酸酶活性切割自身或外源RNA来抑制蛋白合成，抗毒素MazE通过与MazF直接相互作用来抑制其毒性。结核分枝杆菌MazEF家族的九个毒素蛋白中(MazF-mt1到mt9)，MazF-mt3/mt6/mt9切割的底物除了mRNA外、还包括rRNA和tRNA上的特异性位点，并且与结核杆菌对哺乳动物的毒力和耐药性密切相关。本课题以这三个毒素蛋白为研究对象，依托上海和北京同步辐射光源高能量X-射线，解析它们与不同类型底物RNA片段或核糖体组成的复合物三维结构。在此基础上将深入探索蛋白突变体在体内和体外的酶活变化、与底物的相互作用及对宿主生理状态影响，揭示这三个同源蛋白识别底物RNA特异位点和调控蛋白质合成的分子机制。研究成果将为开发以该系统为靶标的新型抗生素和临床耐药性的治理提供参考。

细菌的毒素-抗毒素系统与其耐药性和程序性细胞死亡等多项重要生理过程密切相关，对它们的三维结构和功能深入研究将有助于开发以这些系统作为作用靶点的新型抗菌肽或药物。本课题开展了结核分枝杆菌的重要毒素-抗毒素系统MazF-MazE中的毒素蛋白MazF-mt3/mt6/mt9分别与不同底物RNA的结构和功能研究。我们成功解析了MazF-mt3单体、MazF-mt6单体和MazF-mt3-RNA复合物的晶体结构，并进行了结构分析和相应的生化功能研究，揭示了MazF-mt3特异性识别和切割底物RNA的分子机制。在本课题的资助下我们还对几个与细菌耐药性相关的毒素-抗毒素系统开展了结构和功能研究。通过解析绿脓杆菌的抗毒素蛋白HigA与启动子DNA复合物的结构和蛋白-DNA相互作用研究，揭示了HigA调控毒素-抗毒素系统HigA-HigB的转录以及多个毒力因子合成的新颖分子机制。通过对希瓦氏菌新型毒素-抗毒素系统SO_3165/SO_3166复合物的结构和功能研究，揭示了该复合物的独特结构及新颖的毒素中和机制。以上这些重要的毒素-抗毒素结构和功能研究为开发新型抗菌药物提供了新靶点和重要参考。