Composition and function of the DctA/DcuS sensor complex of E. coli

由膜结合的传感激酶和细胞质反应调节剂组成的双组分系统代表了细菌感知环境刺激的主要装置。许多膜结合传感器激酶需要辅助蛋白或独立蛋白如转运蛋白才能发挥功能。转运蛋白在控制传感器中的分子功能尚不清楚。E.大肠杆菌需要转运蛋白DctA或DcuB来发挥功能。在不存在转运蛋白的情况下，DcuS永久处于活性状态，即使没有效应物富马酸盐。因此，转运蛋白将DcuS转移到对代谢物敏感的状态。准备工作表明：（a）DctA与DcuS形成传感器复合物（DctA/DcuS），（B）DctA通过细胞质螺旋（H8 B）与DcuS相互作用，（c）刺激感知仅通过DcuS而不是复合物中的DctA实现，（d）DctA不是流量传感器，因为转运缺陷变体仍然形成功能性DctA/DcuS传感器复合物。根据现有的函数模型，将传感器DctA由结构变化转化为主管形式。前期工作表明，DcuS的跨膜信号传递依赖于跨膜螺旋2（TM 2）的活塞型易位。TM 2在激活状态时位于周质位置，在非激活状态时位于胞质位置。在建立载体控制的传感激酶研究体系后，（i）DctA/DcuS复合物的组成和DctA-DcuS相互作用的位点，（ii）DctA（或DcuB）对DcuS结构和功能的影响将被表征。DctA/DcuS复合物尚未分离。对于（i），复合物的组成应通过新的质谱分析程序、DctA与DcuS的定义交联以及相互作用肽的鉴定进行研究。为了研究相互作用的机制，将绘制DcuS和DctA的相互作用区域和结构域。对于（ii），DcuS的TM 2的位置将在E.大肠杆菌缺乏DctA或DcuB，或仅含有DcuB的调节缺陷变体。这将显示DctA（或DcuB）是否通过影响TM 2的位置来控制DcuS的功能，并将其转换为实际工作模型预测的基态。除了DctA之外，还将部分通过DcuB进行实验，因为一些实验可以用DcuB以更明确的方式进行，并且因为DcuB（但不是DctA）的明确定义的调控缺陷变体是可用的。

Two-component system consisting of a membrane-bound sensor kinase and a cytoplasmic response regulator represent the major devices of bacteria for sensing environmental stimuli. Many membrane-bound sensor kinases require accessory proteins or independent proteins like transporters for function. The molecular function of the transporters in controlling the sensors is not known. The sensor kinase DcuS (C4-dicarboxylate uptake sensor) of E. coli requires the transporters DctA or alternatively DcuB for function. In the absence of the transporters is DcuS permanent in the active state, that is even without the effector fumarate. The transporters thus transfer DcuS to the fumarate-responsive state. Preparatory work showed that (a) DctA forms a sensor complex with DcuS (DctA/DcuS), (b) DctA interacts by a cytoplasmic helix (H8b) with DcuS, (c) stimulus perception is effected only via DcuS but not by DctA in the complex, and (d ) DctA is no flux sensor since transport deficient variants still form functional DctA/DcuS sensor complexes. According to the present model of function, transforms DctA the sensor DcuS by structural changes to the competent form. Preparatory work showed that transmembrane signal transfer by DcuS relies on a piston-type translocation of transmembrane helix 2 (TM2). TM2 is in the periplasmic position when it is in the activated state and in the cytoplasmic position when it is in the inactive state. Methods for differentiation both positions have been developed.After establishing a system for studying carrier-controlled sensor kinases, (i) the composition of the DctA/DcuS complex and the site of DctA-DcuS interaction, and (ii) the effect of DctA (or DcuB) on structure and function of DcuS shall be characterized. The DctA/DcuS complex was not isolated so far. For (i), the composition of the complex shall be studied by new procedures of mass spectrometry, defined cross-linking of DctA with DcuS and identifying of the interacting peptides. For studies of the mechanism of interaction, the interacting areas and domains of DcuS and DctA will be mapped. For (ii), the position of TM2 of DcuS will be determined in variants of E. coli lacking either DctA or DcuB, or contain only regulatory deficient variants of DcuB. This will show if DctA (or DcuB) control the function of DcuS by affecting the position of TM2 and shift it to the ground state as predicted by the actual working model. The experiments will be performed in part by DcuB in addition to DctA since some experiments can be done in a more clear-cut manner with DcuB, and since well-defined regulation deficient variants of DcuB (but not of DctA) are available.