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ID: 473576.0, MPI für Biophysik / Abt. Molekulare Membranbiologie
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
Authors:Olkhova, Elena; Kozachkov, Lena; Padan, Etana; Michel, Hartmut
Language:English
Date of Publication (YYYY-MM-DD):2009
Title of Journal:Proteins: Structure, Function, and Bioinformatics
Journal Abbrev.:Proteins: Struct., Funct., Bioinf.
Volume:76
Issue / Number:3
Start Page:548
End Page:559
Review Status:Peer-review
Audience:Experts Only
Abstract / Description: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 it's 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 pKa values of Glu78 making them insensitive to pH. Although in the variant D163N the pKa 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 KM. 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
Free Keywords:NhaA Na+/H+ antiporter; conformation; single mutations; pH-dependent activation; continuum electrostatics
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für Biophysik/Abteilung Molekulare Membranbiologie
External Affiliations:Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
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