Molecular Mechanism of Na⁺/H⁺ Antiporting in NhaA

Sodium-proton antiporter NhaA of Escherichia coli is a paradigm to investigate the mechanistic basis of the fundamental Na⁺/H⁺ exchange in cells. However, all existing crystal structures of NhaA are inward-facing (IF), and the putative outward-facing (OF) structures are still unsolved by experiment, limiting a complete understanding of the transport cycle in which Lys300 plays a key role in both structural stability and transport function. Here, we report a set of atomistic molecular dynamics (MD) simulations that start from the structure predicted by an artificial intelligence model that generates function-relevant alternative conformations. It is found that NhaA rapidly relaxes into either the IF or the OF conformation. Furthermore, the neutralization of Lys300 allows the binding of two sodium ions, a configuration that is associated with enhanced conformational sampling. Based on these observations, we propose a sodium-coupled mechanism of Na⁺/H⁺ antiporting.