Structural Analysis of the Escherichia coli KdpFABC Holoenzyme and its Funktional Domains

大肠杆菌KdpFABC全酶的结构分析及其功能域

• 批准号: 5308956
• 负责人: Professor Dr. Horst Kessler
• 金额: --
• 依托单位: Institute for Advanced Study (IAS)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2001
• 资助国家: 德国
• 起止时间: 2000-12-31 至 2004-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5308956?language=en
• 关键词: Structural; Analysis; Escherichia; coli; KdpFABC

In the case of changes in turgor across the cell membrane of Escherichia coli, the cell induces the high-affinity K+-translocating KdpFABC complex, a P-type transport ATPase (P-ATPase) of unique subunit composition and structure. In order to study structural and functional modules of this transport system, the KdpFABC complex as well as functionally important, but soluble domains of KdpB (catalytic subunit) and KdpC should be crystallized and the structures should be solved by X-ray crystallography. The dynamics (ATP binding, phosphorylation, dephosphorylation) and the interplay of the soluble domains, which are now available in homogeneous form, should be studied in solution by NMR. From these studies important information can be obtained about the conformational changes occurring during the reaction cycle of the enzyme complex. The expression of the kdpFABC operon is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. The communication between these two regulatory proteins occurs via phosphorylation and dephosphorylation reactions. Both proteins, as well as a functionally important, but soluble domain of KdpD, should be crystallized and their structures should be solved by X-ray crystallography. Crystals of KdpE have already been obtained. Therefore, the continuation of this part of the project will be given priority. Since the regulatory proteins involved in environmental signalling all exhibit a similar structure (modular design), the information obtained for KdpD and KdpE are pertinent for the understanding of the signal transduction mechanism at the molecular level.

在大肠杆菌细胞膜膨胀度发生变化的情况下，细胞诱导高亲和力的K+-转位KdpFABC复合体，这是一种具有独特亚基组成和结构的P型转运型ATPase(P-ATPase)。为了研究这个运输系统的结构和功能模块，KdpFABC复合体以及KdpB(催化亚单位)和KdpC的重要但可溶的结构域应该结晶，结构需要用X射线结晶学来解决。动力学(ATP结合、磷酸化、去磷酸化)和可溶性结构域的相互作用，现在都是以均一的形式存在的，应该在溶液中用核磁共振研究。从这些研究中，可以获得关于酶复合体在反应周期中发生的构象变化的重要信息。KdpFABC操纵子的表达受膜结合的传感器激酶KdpD和可溶性反应调节因子KdpE的调控。这两种调节蛋白之间的交流通过磷酸化和去磷酸化反应进行。这两种蛋白质以及KdpD的一个重要的但可溶的结构域都应该被结晶，其结构应该通过X射线结晶学来解决。已经获得了KdpE的晶体。因此，这部分项目的延续将被优先考虑。由于参与环境信号转导的调节蛋白都具有相似的结构(模块化设计)，因此KdpD和KdpE的信息对于在分子水平上理解信号转导机制具有重要意义。