title: Understanding the structural requirements for activators of the Kef bacterial potassium efflux system.
abstract: 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.
authors.affiliation: Department of Chemistry, Chemistry Research Laboratory, University of Oxford , Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Jessica
authors.initials: J
authors.lastname: Healy
authors.forename: Silvia
authors.initials: S
authors.lastname: Ekkerman
authors.forename: Christos
authors.initials: C
authors.lastname: Pliotas
authors.forename: Morgiane
authors.initials: M
authors.lastname: Richard
authors.forename: Wendy
authors.initials: W
authors.lastname: Bartlett
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Grayer
authors.forename: Garrett M
authors.initials: GM
authors.lastname: Morris
authors.forename: Samantha
authors.initials: S
authors.lastname: Miller
authors.forename: Ian R
authors.initials: IR
authors.lastname: Booth
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Conway
authors.forename: Tim
authors.initials: T
authors.lastname: Rasmussen
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D008024:Ligands; D011188:Potassium / Q000737:chemistry* / Q000378:metabolism; D027961:Potassium-Hydrogen Antiporters / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020592:Shewanella / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D027961:Potassium-Hydrogen Antiporters; D013388:Succinimides; C421962:kefB protein, E coli; C064530:N-ethylsuccinimido-S-glutathione; D005978:Glutathione; C013585:S-lactoylglutathione; D011188:Potassium