Structures of the ON and OFF states of the membraneous fumarate/Citrate sensor kinases DcuS from E. coli and CitA from Geobacillus thermodenitrifcans

来自大肠杆菌的膜富马酸/柠檬酸传感器激酶 DcuS 和来自热脱硝地芽孢杆菌的 CitA 的 ON 和 OFF 状态的结构

基本信息

批准号：
396465415
负责人：
Professor Dr. Christian Griesinger
金额：
--
依托单位：
Mikrobiologie und Weinforschung
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/396465415?language=en
关键词：
Structures OFF states membraneous fumarate

项目摘要

Sensor kinases (SK) represent a major device of bacteria for responding to extracellular stimuli, resulting in autophosphorylation of the SK. Their function includes signal perception, transmembrane (TM) signaling, and signal transmission to the kinase. Domain composition and transmembrane signaling of the SK show broad variation. The proposal aims at solving the structure of a SK of the DcuS/CitA family (fumarate and citrate sensing) in the ON and the OFF state, and to understand restructuring of the domains in transmembrane and intracellular signaling. DcuS/CitA contain an archaetypical domain composition for transmembrane signaling SK. From the vast number of TM sensor kinases, the structure of the NarQ nitrate SK has been solved in a form depleted of the cytosolic GAF and kinase domains recently. NarQ represents a unique SK with a domain composition largely different from DcuS/CitA and stands very likely for a different mode of signal transduction. DcuS of E. coli and the closely related CitA of the thermophilic Geobacillus thermodenitrificans are homodimers each, composed of an extracytoplasmic PASP (Per ARNT Sim) domain for fumarate or citrate binding, two TM helices (TM1, TM2), a cytoplasmic PASC and the kinase domain. Studies are performed with DcuS or CitA complementarily according to availability of the proteins for the analytic systems. Structures of PASP and PASC have been solved in isolated and membrane-integral constructs in the ON and OFF states. Structural, genetic and biochemical studies show a compaction of PASP upon substrate binding that triggers a piston-like movement of TM2 which is responsible for transmembrane signaling. PASC responds to the shift of TM2 in a structural rearrangement at an alpha-helical dimerization site. This work is the basis for high resolution analysis and an integrated view of the structural rearrangements in the domains during transmembrane signaling. Work will concentrate on (i) on the structural rearrangements in the TM region with the piston-like movement of TM2 that may be combined with a scissors movement of TM2, (ii) the structural rearrangement in PASc during signal transmission and control of the kinase domain. For a complete view and high resolution, structural (NMR and crystallization; AG Griesinger), biochemical, genetic (AG Unden) and biophysical (AG Hinderberger, Griesinger) methods will be applied. Preparatory work established all constructs and the methods for the study, and we presume that DcuS/CitA represent one of the very few SK whose transmembrane and cytoplasmic signaling is within reach at high structural and functional resolution by a combined approach from structural biology, biochemistry and biophysics.

传感器激酶 (SK) 是细菌响应细胞外刺激的主要装置，导致 SK 自身磷酸化。它们的功能包括信号感知、跨膜 (TM) 信号传导以及向激酶的信号传递。 SK 的结构域组成和跨膜信号传导显示出广泛的变化。该提案旨在解决 DcuS/CitA 家族（富马酸和柠檬酸传感）的 SK 在 ON 和 OFF 状态下的结构，并了解跨膜和细胞内信号传导域的重组。 DcuS/CitA 包含跨膜信号 SK 的典型结构域组成。最近，从大量的 TM 传感器激酶中，NarQ 硝酸酯 SK 的结构已以耗尽胞质 GAF 和激酶结构域的形式得到解决。 NarQ 代表一种独特的 SK，其结构域组成与 DcuS/CitA 有很大不同，并且很可能代表不同的信号转导模式。大肠杆菌的 DcuS 和嗜热地芽孢杆菌热脱氮菌的密切相关的 CitA 都是同源二聚体，由用于富马酸或柠檬酸结合的胞质外 PASP (Per ARNT Sim) 结构域、两个 TM 螺旋 (TM1、TM2)、胞质 PASC 和激酶结构域。根据分析系统的蛋白质可用性，使用 DcuS 或 CitA 进行补充研究。 PASP 和 PASC 的结构已在 ON 和 OFF 状态下的分离结构和膜整体结构中得到解决。结构、遗传和生化研究表明，PASP 在底物结合时压缩，触发 TM2 的活塞式运动，TM2 负责跨膜信号传导。 PASC 对 TM2 在 α 螺旋二聚位点的结构重排中的变化做出反应。这项工作是高分辨率分析和跨膜信号传导过程中结构域结构重排的综合视图的基础。工作将集中在 (i) TM 区域的结构重排，TM2 的活塞式运动可能与 TM2 的剪刀运动相结合，(ii) PASc 在信号传输和激酶控制过程中的结构重排领域。为了获得完整的视图和高分辨率，将应用结构（NMR 和结晶；AG Griesinger）、生物化学、遗传（AG Unden）和生物物理（AG Hinderberger、Griesinger）方法。准备工作确定了研究的所有构建体和方法，我们推测 DcuS/CitA 代表了极少数 SK 之一，其跨膜和细胞质信号传导可以通过结构生物学、生物化学和生物化学的组合方法以高结构和功能分辨率实现。生物物理学。