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

Manish Dwivedi, Shahar Sukenik, Assaf Friedler, Etana Padan*

*Corresponding author for this work

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14 Scopus citations


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.

Original languageAmerican English
Article number23339
JournalScientific Reports
StatePublished - 29 Mar 2016

Bibliographical note

Funding Information:
EP and AF thank the DIP (DFG, German-Israeli Project Cooperation # LA3655/1-1). EP and AF thank the Israel Science Foundation (ISF, grant No. 284/12 and No. 939/14, respectively). MD thanks the PBC, Council for Higher Education and the Hebrew University Program for Fellowships for Outstanding Postdoctoral Fellows from China and India (2015).


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