title: Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights.
abstract: Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA.
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, England, UK.
authors.forename: Chiara
authors.initials: C
authors.lastname: Lee
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, England, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England, UK.
authors.forename: Shoko
authors.initials: S
authors.lastname: Yashiro
authors.affiliation: Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: David L
authors.initials: DL
authors.lastname: Dotson
authors.affiliation: Department of Biochemistry and Biophysics, Centre for Biomembrane Research, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Povilas
authors.initials: P
authors.lastname: Uzdavinys
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, England, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Oxford, Didcot, Oxfordshire OX11 0FA, England, UK Japan Science and Technology Agency, ERATO, Human Crystallography Project, Sakyo-ku, Kyoto 606-851, Japan Department of Cell Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, England, UK.
authors.forename: Mark S P
authors.initials: MS
authors.lastname: Sansom
authors.affiliation: Department of Biochemistry and Biophysics, Centre for Biomembrane Research, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Christoph
authors.initials: C
authors.lastname: von Ballmoos
authors.affiliation: Department of Physics, Arizona State University, Tempe, AZ 85287 Department of Biochemistry, University of Oxford, Oxford OX1 3QU, England, UK obeckste@asu.edu ddrew@dbb.su.se a.cameron@warwick.ac.uk.
authors.forename: Oliver
authors.initials: O
authors.lastname: Beckstein
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, England, UK Department of Biochemistry and Biophysics, Centre for Biomembrane Research, Stockholm University, SE-106 91 Stockholm, Sweden obeckste@asu.edu ddrew@dbb.su.se a.cameron@warwick.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Drew
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, England, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Oxford, Didcot, Oxfordshire OX11 0FA, England, UK School of Life Sciences, University of Warwick, Coventry CV4 7AL, England, UK obeckste@asu.edu ddrew@dbb.su.se a.cameron@warwick.ac.uk.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Cameron
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D003460:Crystallization; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D012964:Sodium / Q000737:chemistry*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000648:ultrastructure*; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium