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

Abstract

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.

Figure 1.

Molecular structure of Na⁺/K⁺-ATPase. A, the α-subunit is shown in gray with its cytoplasmic domains colored as follows: A-domain, red; N-domain, blue; and P-domain, green. The β-subunit is shown in violet, and the γ-subunit is shown in orange. B, a superposition of the binding site for sodium-bound E1P (yellow) and potassium-bound E2P (green). C and D, density of ions sampled in 200-ns simulations (blue mesh; all ionizable binding site residues are unprotonated) superimposed on crystal structures for potassium (C) and sodium (D) where the green and yellow spheres represent the position of potassium and sodium ions, respectively, found in published Na⁺/K⁺-ATPase structures. E, a two-dimensional PMF calculated from metadynamics simulations for one potassium ion in the E2P binding site when Asp⁹²⁶ is protonated. Approximate location of sites I, II, and III are highlighted with circles. F, scheme representing the collective variables used to calculate the PMF (see “Experimental procedures”). Black spheres show the reference atoms (α-carbons) used for the metadynamics simulation.

Figure 2.

FEP calculations of relative free energies of binding site protonation states for the sodium-bound E1P (A) and potassium-bound E2P (B) conformations of the Na⁺/K⁺-ATPase are shown. Protonated residues are labeled for each state and indicated by a letter “H” in each schematic. n.d., not determined. The inset in B is a reminder that Asp⁹²⁶ is protonated in all these protonation states (see text). One, Two, Three, or Four Protons denote that one, two, three, or four acidic residues in the binding site are protonated, respectively. C and D, blue circles show the relative free energy of each protonation state obtained from the corresponding top panels with error bars (see “Experimental Procedures” for error bar calculation approach). Red stars show, for each protonation state, the change in free energy incurred about transforming three sodium ions into three potassium ions (C) and two potassium ions to two sodium ions (D).

Figure 3.

Identification of a putative cytoplasmic anion-binding site for Na⁺/K⁺-ATPase. A, average electrostatic potential due to the protonation of key residues in the Na⁺/K⁺-binding site for both the E1P and E2P states. For each binding site residue, the bars show the electrostatic potential calculated using the entire system (yellow) and the subset of protein atoms (red) without considering contributions from that specific residue (see “Experimental procedures”). Error bars denote standard deviations. B, comparison of the relative contributions of lipids, water, solvent cations (K⁺), and anions (Cl⁻) to the electrostatic potential of key binding residues in the E2P conformation. In each case, the maximum electrostatic potential values are subtracted to properly compare the relative contributions of each component (see supplemental Table S12 for values before substraction). C, molecular representation of the cytoplasmic anion-binding site in Na⁺/K⁺-ATPase. Only the α-subunit is shown for clarity. The bound chloride ion is shown as a magenta sphere. D, the distance between the TM6-TM7 loop and the TM8-TM9 loop (top panel) and chloride ion density near the cytoplasmic anion-binding site (lower panel) are compared between the E1P and E2P states. The distance is calculated using the C^δ atom of Glu⁸²¹ and backbone nitrogen of Arg⁹³³. The chloride density is calculated by counting the number of chloride ions within 7 Å of the backbone nitrogen of Arg⁹³³. E, a cavity (blue-gray) extends from Asp⁹²⁶ (shown in “green” licorice) to the cytoplasm in the E2P conformation (right) but is blocked in the E1P state (left). The program fpocket (51) was used for cavity calculations (see “Experimental procedures”). F, schematic representation of a proposed mechanism of TM6-TM7 lid opening, leading to the binding of a chloride ion, protonation of Asp⁹²⁶, and protonation rearrangement of other binding residues.