STRUCTURAL VIEW OF RESPONSE REGULATOR ACTIVATION, NARL

二组分调控系统在细菌中普遍存在，为抗菌药物的开发提供了潜在的靶点。在分子水平上了解膜结合的传感器激酶如何响应输入信号自动磷酸化，选择性地结合和磷酸化其反应调节器，以及激活的反应调节器随后引起适当的细胞功能，是工程药物干预的核心。重点是反应调节因子；我们建议通过应用高分辨核磁共振技术来确定受体结构域磷酸化事件导致反应调节因子NarL的DNA结合结构域激活的结构机制。这将通过首先解决分离的接收器和DNA结合域的结构来实现，我们已经展示了这些结构域在溶液中保持结构化。我们还将确定在磷酸化接收器结构域、将目标DNA与DNA结合结构域结合以及在反式中添加这两个结构域所涉及的结构变化。我们的最终目标将是比较全长NarL的静止态和活跃态的完整的核磁共振结构。

Two-component regulatory systems are ubiquitous in bacteria and offer potential targets for the development of antibacterial drugs. A molecular level understanding of how a membrane bound sensor kinase autophosphorylates in response to an input signal, selectively binds and phosphorylates its response regulator, and the activated response regulator subsequently elicits the appropriate cellular function, is central to engineering pharmaceutical interventions. Focusing on the response regulator; we propose to determine the structural mechanism by which the event of receiver domain phosphorylation results in the activation of the DNA binding domain of the response regulator NarL through the application of high resolution NMR techniques. This will be accomplished by first solving the structures of the isolated receiver and DNA binding domains which we have shown, remain structured in solution. We will also determine the structural changes involved in phosphorylating the receiver domain, binding target DNA to the DNA binding domain, and adding the two domains in trans. Our ultimate goal will be to compare the complete NMR structures of the resting state and the active state of full length NarL.