Understanding the structural requirements for activators of the Kef bacterial potassium efflux system

Jessica Healy, Silvia Ekkerman, Christos Pliotas, Morgiane Richard, Wendy Bartlett, Samuel C. Grayer, Garrett M. Morris, Samantha Miller, Ian R. Booth, Stuart J. Conway, Tim Rasmussen

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)


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, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.

Original languageEnglish
Pages (from-to)1982-1992
Number of pages11
Issue number12
Publication statusPublished - 6 Mar 2014


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