组氨酸激酶HK853的动态ATP lid对反应调节蛋白RR468磷酸活化的影响

Two-component systems (TCS) regulate most important life processes of bacteria, which are potential targets of novel antibacterial drugs. A typical TCS consists of a histidine kinase (HK) and a response regulator (RR), transducing signals through phosphotransfer to trigger cellular regulation. It is not clear how response regulators are activated by receiving phosphoryl group from histidine kinases. Interestingly, we found that the dynamic ATP lid of HK plays an important role in phosphotransfer between HK and RR, but does not affect other functions of HK. In this project, HK853-RR468 was chosen as a model TCS. Low-population transient conformations would be investigated in the form of protein complex by using NMR technology, isotope labeling and protein cross-linking technology. Furthermore, the mechanism of RR activation regulated by ATP lid of HK853 through phosphotransfer would be elucidated in combination with gene mutagenesis and enzymologic assays. In light of the relationships between structure, dynamics and functions, the study would give us a better understanding on the mechanisms of two-component signal transduction systems and provide us theory basis for designing antibacterial drugs to beat back “super bacteria”.

双组分信号转导系统（Two-component System, TCS）调控着细菌的大部分生命活动，是最具潜力的新型抗菌药物靶标之一。细菌的TCS由组氨酸激酶HK和反应调节蛋白RR两部分组成，以磷酸传递的方式实现信号转导从而启动细胞应答。至今尚不明确反应调节蛋白RR如何接受来自组氨酸激酶HK的磷酸基团从而实现活化。我们发现HK上较为动态的ATP lid影响磷酸传递过程却不影响HK的其它功能。本项目拟选用模式蛋白HK853-RR468为研究对象，运用核磁共振动态研究、蛋白质交联、基因突变、同位素标记、酶活测试等技术，在复合物层次上捕捉低丰度瞬态构象并阐述蛋白质结构、动态和功能之间的联系，从原子水平上揭示HK853的ATP lid在磷酸传递过程中对RR活化的作用。该研究将有助于深入理解细菌的信号转导机理，为新型抗菌药物的设计提供理论依据，用以抗击日益严重的“超级细菌”。

双组分信号转导系统 (Two-component system, TCS) 调控细菌大多数重要的生命活动，由组氨酸激酶（Histidine kinase, HK）和应答调节蛋白 (Response regulator, RR) 两部分组成，通过磷酸转移实现信号转导，是重要的新型抗菌药物靶标。磷酸基团如何从组氨酸激酶传递至下游的应答调节蛋白从而实现活化是理解该信号转导机制的关键。本项目针对组氨酸激酶ATP lid如何影响信号转导进行了深入研究。我们综合运用核磁共振技术、X射线晶体衍射技术、原子探针标记技术，酶活测试以及细菌侵染实验等技术手段分别对HK、RR及其复合物，从原子水平、分子水平到细胞水平进行了系统研究，发现了：（1）ATP lid区域的突变影响HK蛋白的自激酶活性，并阐述了木犀草素对HK蛋白的抑制机理；（2）RR蛋白的动态活化机制，并揭示了可能的抑制剂结合位点，同时发展了一种蛋白质翻译后修饰的检测方法；（3）复合物状态下HK行使磷酸酶功能的pH调控分子机制以及酶促反应机理。本项目针对TCS磷酸转移机制的研究为新型抗菌药物的设计提供重要的研究基础。