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

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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

Keywords

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

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    Pliotas, C., Grayer, S. C., Ekkerman, S., Chan, A. K. N., Healy, J., Marius, P., Bartlett, W., Khan, A., Cortopassi, W. A., Chandler, S. A., Rasmussen, T., Benesch, J. L. P., Paton, R. S., Claridge, T. D. W., Miller, S., Booth, I. R., Naismith, J. H., & 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