Mechanistic model of sodium/proton antiport based on X-ray crystal structures and molecular dynamics simulations

Na$^{+}$/H$^{+}$ antiporters are membrane proteins that are vital for cell homeostasis but the mechanistic details of their transport mechanism remain unclear, in particular, how Na$^{+}$ and protons bind to the transporter. We recently solved X-ray crystal structures for two such antiporters (NhaA and NapA) in two different conformations of the transport cycle. All-atom molecular dynamics (MD) simulations (for a total simulated time $>10\ \mu$s), indicate that sodium binding is dependent on the charge states of two conserved aspartate residues. A conserved lysine forms a previously unidentified salt bridge with one of the asparates. Under simulated physiological pH the presence of a Na$^{+}$ ion disrupts and breaks the salt bridge in NhaA. To quantify proton binding, we then performed heuristic p$K_{\mathrm{a}}$ calculations on our ensemble of simulations. The calculations support our novel hypothesis that the conserved lysine in these antiporter binds protons in a sodium-dependent manner and thus acts as part of the transport machinery. In conjunction with simulations of the conformational transition we propose a new mechanistic model of ion binding for the CPA2 class of antiporters within the larger framework of the alternating access mechanism of transmembrane transport.