沙门氏致病菌中组氨酸激酶PmrB结构和信号传递机理

Antibiotic resistance, in particular multi-drug resistance (MDR), is a big threat to current medication against bacterial infections. With more than 100 antibiotics used in clinics, few of them can treat MDR bacteria. Polymyxin, which has been a last resort to fight back many Gram- bacteria, is frequently reported to trigger MDR in several pathogenic bacteria, including Salmonella, Klebsiella, Pseudomonas and Acinetobacter. Polymyxin resistance in these bacteria has been consistently associated with a bacterial two-component system (TCS) PmrAB, which has evolved to become constitutively active under long-term antibiotic treatment by accumulations of mutations. More than 100 mutations, majority of which occur on sensor histidine kinase (SK) component PmrB, have been identified from those clinical MDR specimen. Interestingly, about 80% mutations are found in signal transducer HAMP（Histidine kinases, Adenyl cyclases, Methyl-accepting chemotaxis proteins and Phosphatases）and central DHp domains. Therefore, PmrB is a perfect SK for us to study a long-standing question: how HAMP domain transduces stress signals from extracellular environment to alter DHp and CA activity. We have determined a crystal structure of an entire intracellular region of another SK from streptococcus, called VicK, which includes four domains--HAMP, PAS, DHp and CA. We have also solved crystal structures of VicK homolog at different activation states, and together with molecular dynamic simulations, revealing that DHp domain is a central player for its intrinsic dynamic property. In this proposal, we like to solve a series of crystal structures of PmrB with different conformations of HAMP mutants. With different states of PmrB structures, we will take biochemical, computational and in vivo approaches to dissect their functions. These studies will surely further our understanding of SK-mediated signal transduction pathway and provide a solid ground for new antibiotic development with less MDR potentiality.

双组份系统PmrAB是沙门氏菌等多类致病菌对多粘菌素产生耐药性的分子基础。其组氨酸激酶PmrB 感受多粘菌素信号刺激，通过与跨膜区相连的HAMP结构域将信号传递到下游的DHp和CA结构域。但是，HAMP传递信号的分子机制不明确。我们近期发表了一个带有四个结构域的激酶VicK的晶体结构，提出DHp结构域的不对称性是激酶二聚体顺序完成自激活的关键。我们最近又解析了两个ATP诱导的VicK同源体的晶体结构，揭示了ATP可促进激酶活化态的形成。但这一系列结构没能回答HAMP结构域的调控问题。本研究以PmrB为对象，解析一系列临床上已鉴定带有HAMP的PmrB活化态突变体的晶体结构，综合生物化学，计算机模拟和体内实验等分析结构和功能的关系，揭示HAMP直接调控DHp的分子机制，加强对组氨酸激酶信号转导的理解，促进了组氨酸激酶为靶点的新型抗生素的研发。

双组分系统PmrAB是沙门氏菌等多类致病菌对多粘菌素产生耐药性的分子基础。它的PmrB组氨酸激酶感受抗生素刺激，通过与跨膜区相连的HAMP结构域把信号传递到下游的DHp和CA催化区。我们解析了四个结构域的激酶VicK的晶体结构和两个ATP诱导的VicK同源体的晶体结构，拟在此基础上，研究PmrB的HAMP调控DHp的分子机制。我们从10个细菌中获得了细胞内PmrB蛋白和晶体，得到分辨率5Å，但无法获得HAMP结构信息。因此，我们研究了另个一个重要的双组分系统KdpDE。该系统通过转录调控钾离子通道蛋白亚基来维持胞内的钾离子浓度。它与PmrB一样是双组分系统研究的典型，推断具有类似的信号转导机制。我们已经解析了一个KdpDE复合体结构揭示了该系统信号转导过程，该成果论文在审稿中。另外，我们还解析了一个KdpD与它激活蛋白PtsN的复合体结构，目前正在进行相关的生化实验。本项目的资助有力的支持了我们在双组分系统领域的深入研究，加强了以组氨酸激酶为靶点的新型抗生素的研发基础。