Molecular simulations and free-energy calculations suggest conformation-dependent anion binding to a cytoplasmic site as a mechanism for Na +/K +-ATPase ion selectivity

J Biol Chem. 2017 Jul 28;292(30):12412-12423. doi: 10.1074/jbc.M117.779090. Epub 2017 Jun 6.


Na+/K+-ATPase transports Na+ and K+ ions across the cell membrane via an ion-binding site becoming alternatively accessible to the intra- and extracellular milieu by conformational transitions that confer marked changes in ion-binding stoichiometry and selectivity. To probe the mechanism of these changes, we used molecular simulation and free-energy perturbation approaches to identify probable protonation states of Na+- and K+-coordinating residues in E1P and E2P conformations of Na+/K+-ATPase. Analysis of these simulations revealed a molecular mechanism responsible for the change in protonation state: the conformation-dependent binding of an anion (a chloride ion in our simulations) to a previously unrecognized cytoplasmic site in the loop between transmembrane helices 8 and 9, which influences the electrostatic potential of the crucial Na+-coordinating residue Asp926 This mechanistic model is consistent with experimental observations and provides a molecular-level picture of how E1P to E2P enzyme conformational transitions are coupled to changes in ion-binding stoichiometry and selectivity.

Keywords: Na+/K+-ATPase; anion binding site; free energy perturbation; membrane transport; membrane transporter; molecular dynamics; potassium transport; protonation; selectivity; sodium transport.

MeSH terms

  • Animals
  • Anions / chemistry
  • Anions / metabolism
  • Binding Sites
  • Cytoplasm / chemistry
  • Cytoplasm / metabolism*
  • Models, Molecular
  • Molecular Dynamics Simulation*
  • Protein Conformation
  • Sodium-Potassium-Exchanging ATPase / chemistry*
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Swine
  • Thermodynamics*


  • Anions
  • Sodium-Potassium-Exchanging ATPase

Associated data

  • PDB/2ZXE
  • PDB/3KDP
  • PDB/4HQJ
  • PDB/3WGU