Mechanism of ligand-gated potassium efflux in bacterial pathogens.
Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis.
Drug Development Department, Nevada Cancer Institute, Las Vegas, NV 89135, USA. troosild@nvcancer.org
Tarmo P
TP
Roosild
Samantha
S
Castronovo
Jess
J
Healy
Samantha
S
Miller
Christos
C
Pliotas
Tim
T
Rasmussen
Wendy
W
Bartlett
Stuart J
SJ
Conway
Ian R
IR
Booth
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013388:Succinimides / Q000494:pharmacology
D029968:Escherichia coli Proteins; D008024:Ligands; D050505:Mutant Proteins; D015221:Potassium Channels; D013388:Succinimides; C069287:KefC protein, E coli; C064530:N-ethylsuccinimido-S-glutathione; D005978:Glutathione; D011188:Potassium