Binding Site Protonation and Uphill Occlusion Control the Na+/K+-Pump Selectivity

Abstract

The Na+/K+-pump resides in the plasma membrane and maintains the physiological K+ and Na+ concentration gradient across the cell membrane. It operates via a ping-pong mechanism, making iterative transitions between inward-facing (E1) and outward-facing (E2) conformations. The E1 conformation binds three Na+ from the cytosol and exports them using the energy from ATP hydrolysis. The release of Na+ and the binding of K+ at the extracellular side trigger the structural transition to the occluded E2 state, which imports two K+, as the pump returns to the E1 conformation. Although the general features of the pumping cycle are known, the detailed mechanism governing the selectivity in different states of the pump is not well understood. Using free energy molecular dynamics simulations based on the crystal structures of the pump in its E1 and E2 states, the current study reveals a switch in the binding site protonation state upon the E1-E2 transitions, giving the ion binding site its specificity when the pump is in different states. Ion selectivity of the states along the pump cycle has also been calculated. The relative binding free energy of Na+ and K+ in the binding sites calculated along the conformational transition of the protein reveals a self-correcting mechanism that ensures the selectivity and the stoichiometry of the pumping cycle.