β-sheet-dependent dimerization is essential for the stability of NhaA Na⁺/H⁺ antiporter

A structural model of the NhaA dimer showed that a β-hairpin of each monomer combines to form a β-sheet at the periplasmic side of the membrane. By Cys scanning the entire β-hairpin and testing each Cys replacement for functionality and intermolecular cross-linking, we found that Gln47 and Arg⁴⁹ are critical for the NhaA dimer and that K57C causes an acidic shift of 1 pH unit to the pH dependence of NhaA. Comparing the growth of the NhaA variants with the previously isolated β-hairpin deleted mutant (P45-N58)) and the wild type validated that NhaA dimers have an advantage over monomers in growth under extreme stress conditions and unraveled that during this growth the apparent Km for Na⁺ of P45-N58) was increased 50-fold as compared with the wild type. Remarkably, the effect of the extreme stress on the NhaA variants is reversible. Testing the temperature stability (4-55 °C) of the NhaA variants in dodecyl maltoside micellsshowed that the mutants impaired in dimerization were much less temperature-stable than the wild type. We suggest that NhaA dimers are crucial for the stability of the antiporter under extreme stress conditions.