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Abstract

The crystal structure of NhaA Na⁺/H⁺ antiporter of Escherichia coli has provided a basis to explore the mechanism of Na⁺ and H⁺ exchange and its regulation by pH. However, the dynamics and nature of the pH-induced changes in the proteins remained unknown. Using molecular mechanics methods, we studied the dynamic behavior of the hydrogen-bonded network in NhaA on shifting the pH from 4 to 8. The helical regions preserved the general architecture of NhaA throughout the pH change. In contrast, large conformational drifts occurred at pH 8 in the loop regions, and an increased flexibility of helix IVp was observed on the pH shift. A remarkable pH-induced conformational reorganization was found: at acidic pH helix X is slightly curved, whereas at alkaline pH, it is kinked around residue Lys³⁰⁰. The barrier that exists between the cytoplasmic and periplasmic funnels at low pH is removed, and the two funnels are bridged by hydrogen bonds between water molecules and residues located in the TMSs IV/XI assembly and helix X at alkaline pH. In the variant Gly³³⁸Ser that lost pH control, a hydrogen-bonded chain between Ser³³⁸ and Lys³⁰⁰ was found to block the pH-induced conformational reorganization of helix X.