title: NhaA antiporter functions using 10 helices, and an additional 2 contribute to assembly/stability.
abstract: The Escherichia coli Na(+)/H(+) antiporter (Ec-NhaA) is the best-characterized of all pH-regulated Na(+)/H(+) exchangers that control cellular Na(+) and H(+) homeostasis. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from other antiporters that share the Ec-NhaA structural fold. This α-hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a β-sheet. Here we examine computationally and experimentally the role of the α-hairpin in the stability, dimerization, transport, and pH regulation of Ec-NhaA. Evolutionary analysis (ConSurf) indicates that the VI-VII helical hairpin is much less conserved than the remaining transmembrane region. Moreover, normal mode analysis also shows that intact NhaA and a variant, deleted of the α-hairpin, share similar dynamics, suggesting that the structure may be dispensable. Thus, two truncated Ec-NhaA mutants were constructed, one deleted of the α-hairpin and another also lacking the β-sheet. The mutants were studied at physiological pH in the membrane and in detergent micelles. The findings demonstrate that the truncated mutants retain significant activity and regulatory properties but are defective in the assembly/stability of the Ec-NhaA dimer.
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel; etana@vms.huji.ac.il.
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
authors.affiliation: Protein Expression Facility, Wolfson Centre for Applied Structural Biology, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel;
authors.forename: Tsafi
authors.initials: T
authors.lastname: Danieli
authors.affiliation: Protein Expression Facility, Wolfson Centre for Applied Structural Biology, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel;
authors.forename: Yael
authors.initials: Y
authors.lastname: Keren
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel;
authors.forename: Dudu
authors.initials: D
authors.lastname: Alkoby
authors.affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel;
authors.forename: Gal
authors.initials: G
authors.lastname: Masrati
authors.affiliation: Department of Chemical Engineering, Polymer Research Center, Bebek 34342, Boğaziçi University, Istanbul, Turkey.
authors.forename: Turkan
authors.initials: T
authors.lastname: Haliloglu
authors.affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel;
authors.forename: Nir
authors.initials: N
authors.lastname: Ben-Tal
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel;
authors.forename: Abraham
authors.initials: A
authors.lastname: Rimon
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers
keywords: ConSurf; Na+/H+ antiporter/NhaA; elastic network model; helices truncation; transport protein