Abstract
The Na(+)/K(+)-pump maintains the physiological K(+) and Na(+) electrochemical gradients across the cell membrane. It operates via an 'alternating-access' mechanism, making iterative transitions between inward-facing (E1) and outward-facing (E2) conformations. Although the general features of the transport cycle are known, the detailed physicochemical factors governing the binding site selectivity remain mysterious. Free energy molecular dynamics simulations show that the ion binding sites switch their binding specificity in E1 and E2. This is accompanied by small structural arrangements and changes in protonation states of the coordinating residues. Additional computations on structural models of the intermediate states along the conformational transition pathway reveal that the free energy barrier toward the occlusion step is considerably increased when the wrong type of ion is loaded into the binding pocket, prohibiting the pump cycle from proceeding forward. This self-correcting mechanism strengthens the overall transport selectivity and protects the stoichiometry of the pump cycle.
Highlights
The Na+/K+-pump is a primary active membrane transporter present in most animal cells
In the Na+/K+-pump protonation is exploited to modulate selectivity by altering the electrostatic properties of several of residues in the binding pocket. This is consistent with the results from our previous study on the ion selectivity in E2(K2), which concluded that changes in the electrostatic properties of the protonatable residues was the likely mechanism responsible for the K+ selectivity in the E2 state of the pump (Yu et al, 2011)
Empirical pKa and Free energy perturbation (FEP) calculations based on the molecular dynamics (MD) simulation equilibrated structures indicate that the binding pocket glutamates (i.e., E327, E779, and E954) are likely protonated in both Na3.E1Á(ADPÁPi) and E2(K2), previous mutagenesis experiments showed that charge-neutralizing mutation E327Q affects pump function, possibly by altering ion-pump interactions and the kinetics of the occlusion/deocclusion reactions along the pump cycle (Jorgensen et al, 2001; Kuntzweiler et al, 1995; Li et al, 2006; Nielsen et al, 1998)
Summary
The Na+/K+-pump is a primary active membrane transporter present in most animal cells. It belongs to the P-type ATPase family, which utilizes the energy released from ATP hydrolysis to move ions against their concentration gradients across a membrane barrier. The consensus scheme of the pump cycle is known as the ’Albers-Post’ cycle (Albers, 1967; Post et al, 1969). It involves two major conformations, E1 and E2, with inward- and outward-facing ion binding sites, respectively. The presence of the Na+/K+-pump is essential for excitability and secondary active transport
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