High-Resolution Structures and Molecular Dynamics Simulations of Thermus Thermophilus NapA Reveal a Large-Scale Conformational Change for Ion Translocation

Na+/H+ antiporters are vital for maintaining homeostasis in bacterial cells, in particular for survival in high-salt environments. In humans, these transporters are important drug targets, because their dysfunction is linked to a variety of diseases, including cancer and cardiovascular pathophysiology. However, to date the molecular mechanism by which these proteins transport ions remains poorly understood. We recently solved the structure of the archaeal transporter Thermus Thermophilus NapA in an outward-facing conformation where the putative ion binding site is exposed to the cellular exterior [1]. Using disulfide links to trap the protein, we have now obtained an inward-facing structure of NapA, revealing a large conformational change consistent with the alternating access mechanism long proposed for these transporters. We performed molecular dynamics (MD) simulations demonstrating the disulfide-linked structure to be stable in the absence of the links. Simulations of both inward- and outward-facing conformations show that the dimerization domain anchors the protein in the membrane while the core domain translates the binding site 6-7 A in an elevator-like mechanism. Na+ binding is also dependent on protonation states of the conserved aspartates and lysine for both conformations. This binding is consistent with the hypothesis that the conserved lysine plays a direct role in binding protons in Na+/H+ antiporters that transport 2 H+ [2]. Taken together, structural and simulation data for two distinct conformations of the same transporter give an unambiguous view into the molecular mechanism underlying Na+/H+ antiport.[1] Lee et. al. Nature, 501:573-577, 09 2013. http://dx.doi.org/10.1038/nature12484.[2] Lee et. al. J Gen Physiol, 144:529-544, 2014. http://dx.doi.org/10.1085/jgp.201411219.