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

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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.

Original languageEnglish
Pages (from-to)1982-1992
Number of pages11
JournalBiochemistry
Volume53
Issue number12
Early online date6 Mar 2014
DOIs
Publication statusPublished - 1 Apr 2014

Fingerprint

Potassium
Ligands
Rigid structures
Glutathione
Assays
Lead compounds
Flexible structures
Acidification
Bacteria
Cytoplasm
Hot Temperature
Chemical activation

Keywords

  • Biological Transport, Active
  • Escherichia coli Proteins
  • Glutathione
  • Ion Channel Gating
  • Ligands
  • Potassium
  • Potassium-Hydrogen Antiporters
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Shewanella
  • Succinimides

Cite this

Healy, J., Ekkerman, S., Pliotas, C., Richard, M., Bartlett, W., Grayer, S. C., ... Rasmussen, T. (2014). Understanding the structural requirements for activators of the Kef bacterial potassium efflux system. Biochemistry, 53(12), 1982-1992. https://doi.org/10.1021/bi5001118

Understanding the structural requirements for activators of the Kef bacterial potassium efflux system. / Healy, Jessica; Ekkerman, Silvia; Pliotas, Christos; Richard, Morgiane; Bartlett, Wendy; Grayer, Samuel C; Morris, Garrett M; Miller, Samantha; Booth, Ian R; Conway, Stuart J; Rasmussen, Tim.

In: Biochemistry, Vol. 53, No. 12, 01.04.2014, p. 1982-1992.

Research output: Contribution to journalArticle

Healy, J, Ekkerman, S, Pliotas, C, Richard, M, Bartlett, W, Grayer, SC, Morris, GM, Miller, S, Booth, IR, Conway, SJ & Rasmussen, T 2014, 'Understanding the structural requirements for activators of the Kef bacterial potassium efflux system', Biochemistry, vol. 53, no. 12, pp. 1982-1992. https://doi.org/10.1021/bi5001118
Healy J, Ekkerman S, Pliotas C, Richard M, Bartlett W, Grayer SC et al. Understanding the structural requirements for activators of the Kef bacterial potassium efflux system. Biochemistry. 2014 Apr 1;53(12):1982-1992. https://doi.org/10.1021/bi5001118
Healy, Jessica ; Ekkerman, Silvia ; Pliotas, Christos ; Richard, Morgiane ; Bartlett, Wendy ; Grayer, Samuel C ; Morris, Garrett M ; Miller, Samantha ; Booth, Ian R ; Conway, Stuart J ; Rasmussen, Tim. / Understanding the structural requirements for activators of the Kef bacterial potassium efflux system. In: Biochemistry. 2014 ; Vol. 53, No. 12. pp. 1982-1992.
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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.",
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N2 - 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.

AB - 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.

KW - Biological Transport, Active

KW - Escherichia coli Proteins

KW - Glutathione

KW - Ion Channel Gating

KW - Ligands

KW - Potassium

KW - Potassium-Hydrogen Antiporters

KW - Protein Binding

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Shewanella

KW - Succinimides

U2 - 10.1021/bi5001118

DO - 10.1021/bi5001118

M3 - Article

VL - 53

SP - 1982

EP - 1992

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 12

ER -