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 where Lys300 plays a key role in both structural stability and transport function. Here, we report a set of regular molecular dynamics (MD) simulations that start from the structure predicted by an artificial intelligence method that generates function-relevant alternative conformations. It is found that NhaA rapidly relaxes into either the IF or OF conformation. Further-more, neutralization of Lys300 allows two sodium ions bound to the reaction cavity, which is associated with enhanced conformational sampling. Based on these observations, we propose a sodium-coupled mechanism of Na ⁺ /H ⁺ antiporting.