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.
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 language | English |
|---|---|
| Pages (from-to) | 4219-4234 |
| Number of pages | 16 |
| Journal | Biochemistry |
| Volume | 56 |
| Issue number | 32 |
| Early online date | 28 Jun 2017 |
| DOIs | |
| Publication status | Published - Aug 2017 |
Bibliographical 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.Keywords
- Biophysical studies
- crystallography
- protein dimers
- membrane protein
- nucleotide