title: The Ec-NhaA antiporter switches from antagonistic to synergistic antiport upon a single point mutation.
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.
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Givat Ram, Jerusalem-91904, Israel.
authors.forename: Manish
authors.initials: M
authors.lastname: Dwivedi
authors.affiliation: Institute of Chemistry, the Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem-91904, Israel.
authors.forename: Shahar
authors.initials: S
authors.lastname: Sukenik
authors.affiliation: Institute of Chemistry, the Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem-91904, Israel.
authors.forename: Assaf
authors.initials: A
authors.lastname: Friedler
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Givat Ram, Jerusalem-91904, Israel.
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D008094:Lithium / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D000072417:Protein Domains; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D006859:Hydrogen; D008094:Lithium; D012964:Sodium