Abstract:

The Na⁺, Li⁺/H⁺ antiporter of Escherichia coli (Ec-NhaA) maintains pH, Na⁺ homeostasis in enterobacteria. We used isothermal titration calorimetry to perform a detailed thermodynamic analysis of Li⁺ binding to Ec-NhaA and several of its mutants. We found that, in line with the canonical alternative access mechanistic model of secondary transporters, Li⁺/H⁺ binding to the antiporter is antagonistically coupled. Binding of Li⁺ displaces 2 H⁺from the binding site. The process is enthalpically driven, the enthalpic gain just compensating for an entropic loss and the buffer-associated enthalpic changes dominate the overall free-energy change. Li⁺binding, H⁺ release and antiporter activity were all affected to the same extent by mutations in the Li⁺binding site (D163E, D163N, D164N, D164E), while D133C changed the H⁺/Li⁺ stoichiometry to 4. Most striking, however, was the mutation, A167P, which converted the Ec-NhaA antagonistic binding into synergistic binding which is only known to occur in Cl⁻/H⁺ antiporter.