Recent Structures and Molecular Dynamics Simulations Offer New Perspective on Na+/H+ Antiporters

Abstract

Na þ /H þ antiporters are vital to cells for maintaining homeostasis, especially in high-salt environments. New crystal structures for two such antiporters, cytoplasmic-open Escherichia coli NhaA and periplasmic-open Thermus Thermophilus NapA, show high structural similarity despite low sequence identity. Among their common features are a set of highly conserved charged residues (three aspartates and one lysine) near the putative ion binding site. Using molecular dynamics simulations, we observe that interaction with sodium is dependent on the charge states of the conserved aspartates. The lysine and the nearest asparate also form a previously unidentified salt bridge. Under simulated physiological pH the presence of a sodium ion disrupts and breaks the salt bridge in NhaA. Given the presence of this salt bridge in both protein structures, its behavior under simulation, and the known importance of the conserved lysine, we hypothesize that this salt bridge is directly involved in ion binding and transport. To address the question of sodium binding, we performed an ensemble of all-atom equilibrium molecular dyanamics simulations (over 10 ms in total), varying the protonation states of the conserved residues and quantifying the resulting sodium interaction. To address the question of proton binding, we performed heuristic pKa calculations on this ensemble of simulations, which support the hypothesis that the lysine binds protons in a sodium-dependent manner. In order to elucidate the structural basis for alternating access in NhaA, we created an outward-facing model of NhaA, based on the periplasmic-open NapA structure, and simulated conformational transitions of NhaA from its cytoplasmic- to periplasmic-open state using dynamic importance sampling MD. Taken together, the combination of recent structural and dynamic simulation data suggests a new model of ion binding and transport for the CPA2 class of antiporters. 987-Plat