肺炎链球菌细胞分裂调控蛋白PcsB的结构酶学研究

The most common human pathogen Streptococcus pneumoniae is responsible for many infectious diseases, such as pneumonia, otitis media and meningitis, which lead to over 1.6 million deaths annually in the world. The capsular polysaccharides and the cell-wall degradation fragments are the main pneumococcal virulence factors. Up to date, the detailed molecular mechanisms of the pneumococcal cell-wall divison and remodeling remain largely unknown, which impedes the development of cell wall based vaccines and antibiotics. PcsB (termed after protein required for cell wall separation of group B streptococcus) is not only a cell division regulator, but also a promising vaccine candidate that enters the Phase I clinical trial. Previous studies suggested that the cell division complex FtsEX interacts with PcsB to activate the pneumococcal peptidoglycan hydrolysis and cell-wall remodeling. However, the fine mechanism remains unknown. In this project, we attempt to solve the crystal structures of PcsB in complex with FtsEX or the monoclonol antibody. We will also identify the in vivo interacting partners of PcsB for elucidating the activation mechanism of the peptidoglycan hydrolysis. The findings will provide structural insights into the molecular mechanism of PcsB-regulated peptidoglycan remodeling and pneumococcal cell division, and help the rational design of PcsB-based vaccines against Streptococcus pneumoniae.

肺炎链球菌是人类肺炎、中耳炎、脑膜炎的主要致病菌之一，其感染每年导致全球超过160万人死亡。荚膜多糖和细胞壁降解组分是肺炎链球菌的主要毒力因子，但目前关于其细胞壁分裂和重构的精细分子机制尚不清晰，极大限制了基于细胞壁的疫苗和抗生素的研发。PcsB是一个重要的肺炎链球菌细胞分裂调控蛋白，同时也是目前少数几个进入一期临床的疫苗靶标。PcsB通过与细胞分裂复合物FtsEX的相互作用，激活肽聚糖的水解和细胞壁重构，但具体作用机理不清。本研究将利用X射线晶体学手段解析PcsB蛋白与FtsEX或者单克隆抗体形成的复合物的三维结构，同时寻找并鉴定PcsB的其他相互作用蛋白，建立体内和体外的肽聚糖水解活性检测体系，从而在原子分辨率水平上阐释PcsB参与细胞分裂精细调控的结构酶学基础。同时，基于结构分析鉴定的PcsB抗原决定簇将为PcsB疫苗的设计和优化提供结构生物学指导。

该项目系统研究了细胞分裂蛋白FtsE/X调控PcsB蛋白活性分子机制。通过酶学实验发现FtsEX能够增强PcsB的肽聚糖水解酶活性。我们解析了PcsB的C端的CHAP结构域的1.95 Å的晶体结构。CHAP采用经典的papain折叠模式，并且活性位点具有保守的催化三联体Cys292/His343/Glu360。底物结合口袋分析暗示CHAP可能水解肽聚糖。同时我们解析了FtsEX的同源蛋白Spr0694-0695的3.3 Å的晶体结构，以及Spr0693的3.0 Å的晶体结构， 结构分析发现Spr0693-0694-0695是一个新型的ABC转运蛋白，底物采用侧面接近（lateral access）的方式进入通道转运，其中的一个螺旋调控了底物的进入。