NMR STUDIES OF BACTERIAL SIGNAL TRANSDUCTION

细菌信号转导主要是由双组分系统。 这些系统由两种蛋白质组成，一种是自磷酸化组氨酸激酶，另一种是反应调节剂，它在Mg 2+依赖性反应中通过天冬氨酸残基的磷酸化激活。 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，并且具有高度的同源性，因此双组分系统作为潜在的抗菌剂的新靶点是有吸引力的。 此外，这些系统控制了几种病原生物中毒力和耐药性因子的表达。 双组分途径的抑制可以提供通过用单一抑制剂靶向多种蛋白质来抑制抗性的机会。当磷酸化时，接收器结构域（共振调节器的“开关”组件）调节其同源输出结构域（通常是转录激活结构域）的活性。 由于磷酸-天冬氨酸连接的半衰期短，因此没有获得分离的受体结构域或完整的反应调节剂的磷酸化形式的结构。 本实验室的一个长期目标是使用转录激活因子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，N-末端受体结构域、转录激活结构域和DNA结合结构域。首先，通过NMR确定瞬时磷酸化的接收器结构域的结构。 使用的主要技巧是（a）使用大量过量的磷酸供体产生稳态和（B）添加在多个NMR样品上获得的多个三维数据集。 其次，由磷酸化触发的活化机制将通过NMR弛豫实验和解偶联磷酸化和活化的氨基酸取代来表征。 将通过异源金属离子置换来探测活性位点结构。 第三，将研究从接收器结构域到转录激活结构域的信号级联。 由于全长蛋白质的大小（104 kDa），这个问题具有挑战性。 使用剪接酶内含肽的片段同位素标记方法将与最近开发的NMR技术如TROSY和液晶介质中的偶极偶联相结合。

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.