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

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.