Abstract
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.
Highlights
NhaA is an electrogenic antiporter with a stoichiometry of 2Hϩ/Naϩ [2, 6] and is drastically dependent on pH
Under extreme stress conditions of growth, the wild type dimers were much more beneficial than the mutant monomers in conferring growth resistance [18]. These results have suggested that the -hairpin is needed for NhaA dimerization and that whereas the NhaA monomer is the functional unit of NhaA, the dimers are crucial for growth under extreme stress conditions
The model supported by the crystal structure [17] and cross-linking data [12] revealed two points of contacts between the two NhaA monomers; one, at the cytoplasmic side of the membrane, is formed by few hydrophobic interactions between residues of helices VII and IX, and the other, at the periplasmic side of the membrane, the -sheet are much less stable at high temperature than the is produced by a -sheet formed by joined two -hairpins, one control
Summary
Several main questions have remained open: (i) Which amino acid in the NhaA -hairpin is crucial for maintaining the NhaA dimers? (ii) How is the dimer constructed? These questions are interesting because whereas -sheet is a known common aggregation motif of soluble proteins, oligomerization motifs of membrane proteins are unknown. (iii) Why do the NhaA dimers confer upon the cells higher resistance to Naϩ and Liϩ, as compared with the monomers? To answer these questions, we Cys replaced amino acid residues (each separately and in combination) in the NhaA segment Pro45–Asn, which comprises the -hairpin of NhaA, and studied the mutations with respect to cell growth under various growth conditions, Naϩ/Hϩ antiporter activity at physiological pH, intermolecular cross-linking, aggregation state, and the ability to provide temperature stability to the NhaA molecule in detergent micells and in situ in the membrane. Several main questions have remained open: (i) Which amino acid in the NhaA -hairpin is crucial for maintaining the NhaA dimers? (iii) Why do the NhaA dimers confer upon the cells higher resistance to Naϩ and Liϩ, as compared with the monomers? We Cys replaced amino acid residues (each separately and in combination) in the NhaA segment Pro45–Asn, which comprises the -hairpin of NhaA, and studied the mutations with respect to cell growth under various growth conditions, Naϩ/Hϩ antiporter activity at physiological pH, intermolecular cross-linking, aggregation state, and the ability to provide temperature stability to the NhaA molecule in detergent micells and in situ in the membrane. We found that the NhaA dimers are critically dependent on the -sheet and are crucial for NhaA stability under extreme stress conditions
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