NMR STUDIES OF BACTERIAL SIGNAL TRANSDUCTION

细菌的信号转导主要由双组分系统控制。这些系统由两个蛋白质组成，一个是自动磷酸化的组氨酸激酶，另一个是反应调节因子，在依赖于镁离子的反应中，反应调节因子通过天冬氨酸残基的磷酸化而激活。由于它们对细菌和低等真核生物的生存起着至关重要的作用，并且它们具有很高的同源性，这两个组分系统作为潜在的抗微生物新靶点很有吸引力。此外，这些系统还控制着几种病原生物的毒力和耐药性因子的表达。抑制这两个组分的途径可能提供了一个机会，通过用一个单一的抑制剂靶向多个蛋白质来降低耐药性。当被磷酸化时，接收域(共振调节器的“开关”部分)调节其同源输出域的活性，通常是转录激活域。由于磷酸-天冬氨酸键的半衰期很短，因此没有获得分离的接收域或完整的响应调节器的磷酸化形式的结构。本实验室的一个长期目标是以转录激活因子NTRC(氮调节蛋白C)作为模型系统来阐明反应调节因子的激活机制。NTRC由三个结构域组成，即N-末端接收区、转录激活区和DNA结合区。首先，瞬时磷酸化的接收域的结构将由核磁共振确定。使用的主要技巧是(A)使用大量过量的磷酸盐来创建稳定状态，以及(B)添加在多个核磁共振样品上获取的多个三维数据集。其次，将通过核磁共振松弛实验和将磷酸化和激活解偶联的氨基酸取代来表征由磷酸化触发的激活机制。活性中心结构将通过异种金属离子置换来探测。第三，将研究从接收域到转录激活域的信号级联。由于全长蛋白质(104 KDa)的大小，这个问题具有挑战性。使用剪接酶内含子的分段同位素标记方法将与最近开发的核磁共振技术相结合，如液晶介质中的TROSY和偶极耦合。

Bacterial signal transduction is predominated by two-component systems. These systems consist of two proteins, an autophosphorylating histidine kinase and a response regulator, which is activated by phosphorylation at an aspartate residue in a Mg2+ dependent reaction. Because of their crucial role for the survival of bacteria and lower eukaryotes and its high homology, two component systems are attractive as potential new targets for antimicrobials. In addition, these systems control the expression of virulence and drug resistance factors in several pathogenic organisms. Inhibition of the two component pathway may present an opportunity to depress resistance by targeting multiple proteins with a single inhibitor. When phosphorylated, the receiver domain ("switch" component of the resonse regulator) modulates the activity of its cognate output domain, often a transcriptional activation domain. No structure has been obtained for the phosphorylated form of either an isolated receiver domain or an intact response regulator due to the short half-life of the phospho-aspartate linkage. A long- term goal of this laboratory is to elucidate the mechanism of activation of response regulators using the transcriptional activator NtrC (nitrogen regulatory protein C) as model system. NtrC consists of three domains, the N-terminal receiver domain, the transcriptional activation domain and the DNA-binding domain. First, the structure of the transiently phosphorylated receiver domain will be determined by NMR. The main tricks used are (a) creating a steady state using large excess of phosphodonor and (b) adding multiple three dimensional data sets taken on multiple NMR samples. Second, the mechanism of activation triggered by phosphorylation will be characterized by NMR relaxation experiments and amino acid substitutions that uncouple phosphorylation and activation. The active site structure will be probed by heterologous metal ion replacement. Third, the signal cascade from the receiver domain to the transcriptional activation domain will be investigated. This problem is challenging because of the size of the full-length protein (104 kDa). Methods for segmental isotopic labeling using the splicing enzymes inteins will be combined with recently developed NMR techniques such as TROSY and dipolar couplings in liquid crystalline medium.