The Ec-NhaA antiporter switches from antagonistic to synergistic antiport upon a single point mutation.

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.