Understanding the Structural Requirements for Activatorsof the Kef Bacterial Potassium Efflux System

摘要

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of ∼10–20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10–20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibitsa similar Kef affinity to GSH. Synthetic ligands were synthesizedto contain either rigid or flexible structures and investigated asligands for Kef. Compounds with rigid structures and high affinityactivated Kef. In contrast, flexible ligands with similar bindingaffinities did not activate Kef. These data provide insight into thestructural requirements for Kef gating, paving the way for the developmentof a screen for potential therapeutic lead compounds targeting theKef system.