Beta-sheet-dependent dimerization is essential for the stability of NhaA Na+/H+ antiporter.
A structural model of the NhaA dimer showed that a beta-hairpin of each monomer combines to form a beta-sheet at the periplasmic side of the membrane. By Cys scanning the entire beta-hairpin and testing each Cys replacement for functionality and intermolecular cross-linking, we found that Gln47 and Arg49 are critical for the NhaA dimer and that K57C causes an acidic shift of 1 pH unit to the pH dependence of NhaA. Comparing the growth of the NhaA variants with the previously isolated beta-hairpin deleted mutant (Delta(P45-N58)) and the wild type validated that NhaA dimers have an advantage over monomers in growth under extreme stress conditions and unraveled that during this growth the apparent Km for Na+ of Delta(P45-N58) was increased 50-fold as compared with the wild type. Remarkably, the effect of the extreme stress on the NhaA variants is reversible. Testing the temperature stability (4-55 degrees C) of the NhaA variants in dodecyl maltoside micells showed that the mutants impaired in dimerization were much less temperature-stable than the wild type. We suggest that NhaA dimers are crucial for the stability of the antiporter under extreme stress conditions.
Herz
Katia
K
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Rimon
Abraham
A
Jeschke
Gunnar
G
Padan
Etana
E
D019943:Amino Acid Substitution; D019281:Dimerization; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D055550:Protein Stability; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological / Q000502:physiology*
D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium