Revealing the ligand binding site of NhaA Na+/H+ antiporter and its pH dependence.
pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na(+) nor Li(+), the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li(+) in detergent micelles. This interaction is driven by an increase in enthalpy (ΔH of -8000 ± 300 cal/mol and ΔS of -15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li(+) binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li(+) binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter.
Institute of Chemistry, Alexander Silberman Institute of Life Sciences, Faculty of Sciences, Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
Michal
M
Maes
Abraham
A
Rimon
Lena
L
Kozachkov-Magrisso
Assaf
A
Friedler
Etana
E
Padan
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry / Q000379:methods; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008094:Lithium / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D008024:Ligands; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D008094:Lithium