Control of AmtB-GlnK Complex Formation by Intracellular Levels of ATP, ADP, and 2-Oxoglutarate
Control of AmtB-GlnK Complex Formation by Intracellular Levels of ATP, ADP, and 2-Oxoglutarate
复制标题
DOI：
10.1074/jbc.m110.153908
复制
发表时间：
2010-10-01
影响因子：
4.8
通讯作者：
Merrick, Mike
中科院分区：
文献类型：
--
作者：
Radchenko, Martha V.;Thornton, Jeremy;Merrick, Mike
P-II蛋白是自然界中最广泛的信号转导蛋白家族之一，在细菌、古细菌和植物中普遍存在。它们通过与多种酶、转录因子和膜转运蛋白相互作用并调节其活性，在协调氮代谢中发挥重要作用。P-II蛋白的调节特性来源于它们结合三种效应物的能力：ATP、ADP和2-酮戊二酸。然而，一个明确的模型，整合生理变化与相应的结构变化，介导P-II与靶蛋白的相互作用，迄今尚未开发。在这项研究中，我们分析了在大肠杆菌细胞内效应池的波动，在协会和解离的P-II蛋白GlnK与氨通道AmtB。我们确定，促进AmtB-GlnK复合物形成的关键特征是铵流入后2-酮戊二酸池的快速下降和ATP/ADP比率的同时但短暂的变化。我们还能够使用我们在体内观察到的相同效应器组合在体外复制AmtB-GlnK相互作用。这一全面的数据集使我们能够提出一个模型，解释GlnK及其效应子之间的相互作用如何影响GlnK的构象，从而调节其与AmtB的相互作用。
P-II proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. They play a major role in coordinating nitrogen metabolism by interacting with, and regulating the activities of, a variety of enzymes, transcription factors, and membrane transport proteins. The regulatory properties of P-II proteins derive from their ability to bind three effectors: ATP, ADP, and 2-oxoglutarate. However, a clear model to integrate physiological changes with the consequential structural changes that mediate P-II interaction with a target protein has so far not been developed. In this study, we analyzed the fluctuations in intracellular effector pools in Escherichia coli during association and dissociation of the P-II protein GlnK with the ammonia channel AmtB. We determined that key features promoting AmtB-GlnK complex formation are the rapid drop in the 2-oxoglutarate pool upon ammonium influx and a simultaneous, but transient, change in the ATP/ADP ratio. We were also able to replicate AmtB-GlnK interactions in vitro using the same effector combinations that we observed in vivo. This comprehensive data set allows us to propose a model that explains the way in which interactions between GlnK and its effectors influence the conformation of GlnK and thereby regulate its interaction with AmtB.