链球菌中关键的双组分系统VicRK复合体的结构和信号传导的分子机制

VicK and VicR consist of an essential two-component system (TCS), which is responsible for transcriptional regulations in several pathogens of Gram-positive bacteria, including Streptococcus pneumoniae, Staphylococcus aureus, Baccillus anthracis and Streptococcus mutans. VicK is a Class I sensor histidine kinase (SK). SKs can directly sense a variety of environmental stimuli, such as light, nutrition, osmolarity, toxins and even redox potential. VicR is a transcriptional factor, called response regulator, which binds the promoters of a special set of downstream genes to activate their transcriptions, when induced by phosphorylation of VicK. Supported by NSFC, we have determined a crystal structure of intracellular domains of VicK and discovered a sequential activation mechanism for autokinase activation. In order to further our understanding on sensing and signal transductions, we have been interested in the relationship of VicK and its cognate regulator VicR. We have found that VicK can physically interact with VicR. Interestingly, VicR is competed off by VicK for its specific DNA binding elements. We have obtained some well-diffracting crystals of VicK and VicR complex. Here we propose to dissect the relationship of VicK, VicR and DNA by biochemistry, microbiology and structural biology approaches, and to uncover the molecular mechanisms of signal sensing and transduction by VicRK system. This proposed research could build a stronger base to develop any new antibiotics for controls of those pathogens listed above.

VicK和VicR组成了肺炎链球菌等致病菌中一种生死攸关的双组分系统。VicK是一种感应外界信号的组氨酸蛋白激酶，VicR是一种转录因子，称为感应调节子，控制下游目的基因的表达。我们在前期国家自然科学基金的支持下，已经解析了VicK胞内部分的晶体结构，发现了该激酶激活的新的分子机制，并探讨了它感应和传递信号的可能途径。在此基础上，我们进一步研究了VicK如何激活它的特异调节子VicR。我们发现VicR能与VicK发生直接的相互作用，并竞争性的结合起动子的上游DNA调节元件。我们还获得了具有衍射能力的VicK和VicR复合体的晶体。因此我们计划通过生物化学，微生物学和结构生物学等手段研究VicK、VicR与DNA三者间的关系，并阐明VicK将外界的信号传递到VicR，从而调节下游目的基因表达的分子机理，为利用VicRK这个双组分系统开发对肺炎链球菌等致病菌的新型抗菌素提供理论基础。

VicK／VicR和它的同源体是肺炎pneumonia等致病的革兰氏阳性球菌中生死攸关的双组分系统（two component system，TCS)。VicK是带有信号转导元件HAMP和感应元件PAS的组氨酸激酶。它首先负责对各种胞内外的刺激作出反应，改变自身的磷酸化状态，进一步调节VicR的结合DNA启动子，介导各种应急性基因的转录。几年前，我们幸运地受到国家自然科学基金委的资助，成功地解析了全长的VicK的晶体结构，但没有结合的ATP，分辨率也只有3.1Å。因此这几年来，我们继续解析了VicK同源体和ATP结合的精细结构，看到了ATP与Histidine靠近并形成了氢键，这是自磷酸化的必要条件。 我们还成功的表达了带有跨膜区的全长VicK，并分析了它对离子和pH的反应。我们还对VicK／VicR复合体的结晶进行了初步的探索，获得了低分辨率的晶体。