title: Combined computational and biochemical study reveals the importance of electrostatic interactions between the "pH sensor" and the cation binding site of the sodium/proton antiporter NhaA of Escherichia coli.
abstract: Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. The crystal structure of NhaA has provided a basis to explore the mechanism of ion exchange and its unique regulation by pH. Here, the mechanism of the pH activation of the antiporter is investigated through functional and computational studies of several variants with mutations in the ion-binding site (D163, D164). The most significant difference found computationally between the wild type antiporter and the active site variants, D163E and D164N, are low pK(a) values of Glu78 making them insensitive to pH. Although in the variant D163N the pK(a) of Glu78 is comparable to the physiological one, this variant cannot demonstrate the long-range electrostatic effect of Glu78 on the pH-dependent structural reorganization of trans-membrane helix X and, hence, is proposed to be inactive. In marked contrast, variant D164E remains sensitive to pH and can be activated by alkaline pH shift. Remarkably, as expected computationally and discovered here biochemically, D164E is viable and active in Na(+)/H(+) exchange albeit with increased apparent K(M). Our results unravel the unique electrostatic network of NhaA that connect the coupled clusters of the "pH sensor" with the binding site, which is crucial for pH activation of NhaA.
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany. elena.olkhova@mpibp-frankfurt.mpg.de
authors.forename: Elena
authors.initials: E
authors.lastname: Olkhova
authors.forename: Lena
authors.initials: L
authors.lastname: Kozachkov
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D001665:Binding Sites; D002412:Cations / Q000737:chemistry*; D019295:Computational Biology / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity
chemical_list: D002412:Cations; D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers