Pliotas Christos, Grayer Samuel C, Ekkerman Silvia, Chan Anthony K N, Healy Jess, Marius Phedra, Bartlett Wendy, Khan Amjad, Cortopassi Wilian A, Chandler Shane A, Rasmussen Tim, Benesch Justin L P, Paton Robert S, Claridge Timothy D W, Miller Samantha, Booth Ian R, Naismith James H, Conway Stuart J
Biomedical Sciences Research Complex, University of St Andrews , North Haugh, St Andrews KY16 9ST, U.K.
Department of Chemistry, Chemistry Research Laboratory, University of Oxford , Mansfield Road, Oxford OX1 3TA, U.K.
Biochemistry. 2017 Aug 15;56(32):4219-4234. doi: 10.1021/acs.biochem.7b00300. Epub 2017 Aug 4.
Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.
配体结合是蛋白质最基本的特性之一。配体功能可分为三种基本类型：底物、调节分子以及对蛋白质稳定性、反应活性或酶 - 底物复合物形成至关重要的辅因子。细菌中钾离子运动的调节主要受调节配体的控制，这些配体控制相关通道和转运体，它们拥有包含罗斯曼折叠（RFs）的亚基或结构域。在此，我们证明单磷酸腺苷（AMP）与二聚体细菌Kef钾外流系统（Kef）的两个RFs结合，在其中发挥结构作用。我们得出结论，AMP以高亲和力结合，确保该位点在细胞内始终被完全占据。我们证明，失去结合AMP的能力会导致蛋白质以及可能的二聚体不稳定，进而导致功能丧失。Kef系统功能通过在二聚体亚基之间的界面处相对低亲和力的基于谷胱甘肽的配体的可逆结合来调节。我们提出，这个界面结合位点本身至少部分地通过AMP结合而得以稳定。
