Sodium-proton antiporters are regarded as important drug targets for developing cardiovascular therapeutics. Yet with no human sodium-proton antiporters available, prokaryotic analogs such as NhaA from <i>E. coli</i> act as important model systems. NhaA exchanges two protons for one sodium ion and it’s hypothesized that the two proton binding sites are Asp164 and Lys300. However, mutagenesis studies have cast doubt on the role of Lys300 since mutation of this site demonstrates retained activity. To address this controversy, we performed state-of-the-art continuous constant pH molecular dynamics simulations on NhaA mutants. These simulations suggest the activity of this antiporter is retained through the use of an alternative proton binding site Asp133. Asp133 can act as the sole second proton binding site, seen in K300R, or in conjunction with Asp163 as seen in K300A. In K300Q/D163N, Asp164 and Asp133 jointly bind two protons. Electrostatic coupling between Asp133 and Asp163 or Asp164 is enabled via a network of proton-coupled hydrogen bonds. These data resolve the controversy and highlight the intricacy of a compensatory transport mechanism of NhaA mutants, which may also represent how alternative proton-binding sites are an important general mechanism of proton-coupled transport in secondary active transporters.

Continuous constant pH simulations revealed an alternative proton-binding site and long-distance coupling in an ion-proton antiporter

ACS Spring Meeting 2021

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