bi5001118_si_001.pdf (8.7 MB)
Understanding the Structural Requirements for Activators of the Kef Bacterial Potassium Efflux System
journal contribution
posted on 2015-12-17, 01:19 authored by 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 RasmussenThe 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.