β-Sheet-dependent Dimerization Is Essential for the Stability of NhaA Na+/H+ Antiporter

• Published in: The Journal of biological chemistry Vol. 284; no. 10; pp. 6337 - 6347
• Main Authors: Herz, Katia, Rimon, Abraham, Jeschke, Gunnar, Padan, Etana
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.03.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Dimerization; Escherichia coli K12 - genetics; Escherichia coli K12 - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Hydrogen-Ion Concentration; Mutation; Protein Stability; Protein Structure, Quaternary - physiology; Protein Structure, Secondary - physiology; Sodium - metabolism; Sodium-Hydrogen Exchangers - genetics; Sodium-Hydrogen Exchangers - metabolism; Stress, Physiological - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M807720200

A structural model of the NhaA dimer showed that a β-hairpin of each monomer combines to form a β-sheet at the periplasmic side of the membrane. By Cys scanning the entire β-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 β-hairpin deleted mutant (Δ(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 Δ(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 °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.