Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System

Christos Pliotas, Samuel C Grayer, Silvia Ekkerman, Anthony K. N. Chan, Jess Healy, Phedra Marius, Wendy Bartlett, Amjad Khan, Wilian A. Cortopassi, Shane A. Chandler, Tim Rasmussen, Justin L. P. Benesch, Robert S. Paton, Timothy D. W. Claridge, Samantha Miller, Ian R Booth, James H. Naismith, Stuart J Conway

Research output: Contribution to journalArticle

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Abstract

Ligand binding is one of the most fundamental properties of proteins. Ligand
functions fall into three basic types: substrates, regulatory molecules, and co-factors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that AMP is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Regulation of Kef system function is via the reversible binding of comparatively low affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilised, at least in part, by AMP binding.
Original languageEnglish
Pages (from-to)4219-4234
Number of pages16
JournalBiochemistry
Volume56
Issue number32
Early online date28 Jun 2017
DOIs
Publication statusPublished - Aug 2017

Fingerprint

Shewanella
Adenosine Monophosphate
Potassium
Ligands
Dimers
Proteins
Protein Stability
Substrates
Glutathione
Bacteria
Binding Sites
Ions
Molecules
Enzymes

Keywords

  • Biophysical studies
  • crystallography
  • protein dimers
  • membrane protein
  • nucleotide

Cite this

Pliotas, C., Grayer, S. C., Ekkerman, S., Chan, A. K. N., Healy, J., Marius, P., ... Conway, S. J. (2017). Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System. Biochemistry, 56(32), 4219-4234. https://doi.org/10.1021/acs.biochem.7b00300

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System. / Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K. N. ; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad ; Cortopassi, Wilian A. ; Chandler, Shane A. ; Rasmussen, Tim; Benesch, Justin L. P. ; Paton, Robert S. ; Claridge, Timothy D. W. ; Miller, Samantha; Booth, Ian R; Naismith, James H.; Conway, Stuart J.

In: Biochemistry, Vol. 56, No. 32, 08.2017, p. 4219-4234.

Research output: Contribution to journalArticle

Pliotas, C, Grayer, SC, Ekkerman, S, Chan, AKN, Healy, J, Marius, P, Bartlett, W, Khan, A, Cortopassi, WA, Chandler, SA, Rasmussen, T, Benesch, JLP, Paton, RS, Claridge, TDW, Miller, S, Booth, IR, Naismith, JH & Conway, SJ 2017, 'Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System' Biochemistry, vol. 56, no. 32, pp. 4219-4234. https://doi.org/10.1021/acs.biochem.7b00300
Pliotas, Christos ; Grayer, Samuel C ; Ekkerman, Silvia ; Chan, Anthony K. N. ; Healy, Jess ; Marius, Phedra ; Bartlett, Wendy ; Khan, Amjad ; Cortopassi, Wilian A. ; Chandler, Shane A. ; Rasmussen, Tim ; Benesch, Justin L. P. ; Paton, Robert S. ; Claridge, Timothy D. W. ; Miller, Samantha ; Booth, Ian R ; Naismith, James H. ; Conway, Stuart J. / Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System. In: Biochemistry. 2017 ; Vol. 56, No. 32. pp. 4219-4234.
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abstract = "Ligand binding is one of the most fundamental properties of proteins. Ligandfunctions fall into three basic types: substrates, regulatory molecules, and co-factors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that AMP is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Regulation of Kef system function is via the reversible binding of comparatively low affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilised, at least in part, by AMP binding.",
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note = "The project is supported by the Wellcome Trust (WT092552MA, WT100209MA) to IRB, JHN, SM, and SJC, a Biotechnology and Biological Sciences Research Council grant (BB/H017917/1), and a European Union Marie Curie ITN Award (NICHE; 289384) that supported SE. CP would also like to acknowledge additional support by a Tenovus Scotland grant award (T15/41). WAC is supported by a Science Without Borders scholarship. The authors would like to acknowledge that the work presented here made use of the Emerald High Performance Computing facility made available by the Centre for Innovation, formed by the universities of Oxford, Southampton, Bristol, and University College London in partnership with the STFC Rutherford Appleton Laboratory. RSP acknowledges the use of the EPSRC UK National Service for Computational Chemistry Software (NSCCS) at Imperial College London in carrying out this work (CHEM773). RSP is grateful to NVIDIA for the generous donation of Tesla GPUs as part of the academic partnership scheme. SJC and AC thank the European Commission for the award of a Marie Curie Fellowship to AC (660156, FLUOROKEF). SJC and SCG thank the EPSRC for studentship support forSCG. SJC thanks St. Hugh’s College, Oxford, for research support.",
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T1 - Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System

AU - Pliotas, Christos

AU - Grayer, Samuel C

AU - Ekkerman, Silvia

AU - Chan, Anthony K. N.

AU - Healy, Jess

AU - Marius, Phedra

AU - Bartlett, Wendy

AU - Khan, Amjad

AU - Cortopassi, Wilian A.

AU - Chandler, Shane A.

AU - Rasmussen, Tim

AU - Benesch, Justin L. P.

AU - Paton, Robert S.

AU - Claridge, Timothy D. W.

AU - Miller, Samantha

AU - Booth, Ian R

AU - Naismith, James H.

AU - Conway, Stuart J

N1 - The project is supported by the Wellcome Trust (WT092552MA, WT100209MA) to IRB, JHN, SM, and SJC, a Biotechnology and Biological Sciences Research Council grant (BB/H017917/1), and a European Union Marie Curie ITN Award (NICHE; 289384) that supported SE. CP would also like to acknowledge additional support by a Tenovus Scotland grant award (T15/41). WAC is supported by a Science Without Borders scholarship. The authors would like to acknowledge that the work presented here made use of the Emerald High Performance Computing facility made available by the Centre for Innovation, formed by the universities of Oxford, Southampton, Bristol, and University College London in partnership with the STFC Rutherford Appleton Laboratory. RSP acknowledges the use of the EPSRC UK National Service for Computational Chemistry Software (NSCCS) at Imperial College London in carrying out this work (CHEM773). RSP is grateful to NVIDIA for the generous donation of Tesla GPUs as part of the academic partnership scheme. SJC and AC thank the European Commission for the award of a Marie Curie Fellowship to AC (660156, FLUOROKEF). SJC and SCG thank the EPSRC for studentship support forSCG. SJC thanks St. Hugh’s College, Oxford, for research support.

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N2 - Ligand binding is one of the most fundamental properties of proteins. Ligandfunctions fall into three basic types: substrates, regulatory molecules, and co-factors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that AMP is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Regulation of Kef system function is via the reversible binding of comparatively low affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilised, at least in part, by AMP binding.

AB - Ligand binding is one of the most fundamental properties of proteins. Ligandfunctions fall into three basic types: substrates, regulatory molecules, and co-factors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that AMP is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Regulation of Kef system function is via the reversible binding of comparatively low affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilised, at least in part, by AMP binding.

KW - Biophysical studies

KW - crystallography

KW - protein dimers

KW - membrane protein

KW - nucleotide

U2 - 10.1021/acs.biochem.7b00300

DO - 10.1021/acs.biochem.7b00300

M3 - Article

VL - 56

SP - 4219

EP - 4234

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 32

ER -