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 D926. 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. Na + /K + -ATPase is a membrane protein that actively transports sodium ions out of the cell while importing potassium ions, both against their electrochemical gradients, thus providing potential energy necessary for many essential cellular functions (1-3). Malfunction of Na + /K + -ATPase has been linked to numerous diseases including impaired memory and learning, familial hemiplegic migraine 2, rapid-onset dystonia Parkinsonism, and heart failure (4-6), thus, Na + /K + -ATPase is an important target for treatment of brain and heart conditions (7,8).By harnessing chemical energy stored in ATP, the Na + /K + -ATPase cycles between two major conformational states during active ion pumping: one state with high affinity for sodium ions (E1), and a second with high affinity for potassium ion (E2).While the complete mechanism of function of the Na Na+ /K + -ATPase and ion selectivity 2 more complicated, involving several conformational transitions described more fully in the Post-Albers scheme (9-11), here we focus on the sodium-bond E1P and potassium-bound E2P states (i.e. phosphorylated E1 and E2), for which crystal structures are available (12)(13)(14)(15)(16)(17). One of the central features of ion transport by the Na + /K + -ATPase is a change in ion selectivity between E1 and E2 conformations (3). The origin of this selectivity change has been a focus of Na + /K + -ATPase research since its discovery by Jens Christian Skou in 1957 (1). Extensive mutational studies have identified key residues involved in ion binding, which include five acidic residues: Asp804, Asp808, and Asp926, Glu327 and Glu779 (13) whose orientation, distance, and ion coordination have been proposed to be involved in determining selectivity (3). The first crystal structure of the Na + /K + -ATPase, solved for the potassium-bound E2P state in 2007 (15) and later followed by several higher resolution structures (14,16,17), revealed a ...

Determining the molecular basis of voltage sensitivity in membrane proteins

“…aThe trajectory used for the analysis was taken from Yazdi et al (2016).bThe trajectory for the closed capsaicin-bound state of TRPV1 used for the analysis was taken from Kasimova et al (2018).cThe trajectory for the sodium-bound Na + /K + ATPase used for the analysis was taken from Razavi et al (2017).…”

“…cThe trajectory for the sodium-bound Na + /K + ATPase used for the analysis was taken from Razavi et al (2017).…”

K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase

“…In general, the number of deprotonated residues in the cation binding site will correlate to the number of bound K + , but this can be influenced by other moieties like water molecules and backbone carbonyl groups which can contribute coordinating oxygen atoms to the cation. Furthermore, the distribution of the protonated residues on acidic residues and water molecules can also matter, as has already been speculated for the Na + , K + -ATPase 23 – 26 .…”

A Model for the Homotypic Interaction between Na+,K+-ATPase β1 Subunits Reveals the Role of Extracellular Residues 221–229 in Its Ig-Like Domain

“…It is expected that the β 1 subunit suffers conformational changes in response to changes in α-subunit, during the catalytic cycle [42]. There are reports of MD simulations of the complete Na + , K + -ATPase [43,44]. However, they offer no description of the structural relationship between α and β subunits.…”