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Mechanisms of pH activation and ion translocation in Na + /H + antiporters studied by EM

Mechanisms of pH activation and ion translocation in Na + /H + antiporters studied by EM. Werner Kühlbrandt Max-Planck-Institut für Biophysik Frankfurt am Main Erice 16-6-06. Na + /H + antiporters. Secondary transporters Ubiquitous in all cells (animals, plants, prokaryotes)

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Mechanisms of pH activation and ion translocation in Na + /H + antiporters studied by EM

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  1. Mechanisms of pH activation and ion translocation in Na+/H+ antiporters studied by EM Werner Kühlbrandt Max-Planck-Institut für Biophysik Frankfurt am Main Erice 16-6-06

  2. Na+/H+ antiporters • Secondary transporters • Ubiquitous in all cells (animals, plants, prokaryotes) • Maintain intracellular pH and Na+ concentration • pH-activated • E. coli Na+/H+ antiporter NhaA • Mammalian Na+/H+ exchanger NHE1 • M. jannaschii Na+/H+ antiporter MjNhaP1

  3. NhaA: Na+/H+ antiporter A • Family prototype from E. coli • Well-characterized • 3.5 Å X-ray structure known • Maintains intracellular pH and Na+ concentration • Adaptation to high salinity, high pH • Inactive below pH6 • Activated at pH7 • Fully active at pH8

  4. Tubular crystals of NhaA

  5. 2D crystals of E.coli NhaA 1 µm

  6. 3D map of E. coli NhaA dimerat 7 Å resolution A (Williams, Nature 2000)

  7. X-ray structure of NhaA monomer at 3.45 Å resolution (Hunte et al, Nature 2005)  hairpin periplasmic view cytoplasmic view

  8. 2. MjNhaP1Na+/H+ antiporter from M. jannaschiiHomologue of human NHE-1 antiporter and E. coli NhaAfrom hyperthermophilic archaeon

  9. 2D crystals of M. jannaschii NhaP1 100 nm 1 µm Tubular crystals

  10. NhaP1 projection data 15Å 10Å 8Å 6Å

  11. NhaP1 8Å projection map at pH 4

  12. NhaP1 8Å projection map atpH 8

  13. NhaP1 pH4 - pH 8 difference map

  14. NhaP1 pH4 - pH 4difference map

  15. Comparison of Na+/H+ antiporters MjNhaP1 E. coli NhaA pH 4 pH 8 (Williams et al, EMBO J. 1999) (Vinothkumar et al, EMBO J. 2005)

  16. pH induced helix movements in MjNhaP1 dimer helix bundle dimer interface helix bundle

  17. pH-dependent activity of MjNhaP1 in liposomes MjNhaP1 liposomes, 0.3 M NaCl inside empty liposomes (control) pH 6 no NaCl gradient acridine orange fluorescence (arbitrary units) pH 7.5 add liposomes 0.6 min NH4Cl add NH4Cl

  18. Implications for pH activation and ion transport in MjNhaP1 (Vinothkumar et al, EMBO J. 2005)

  19. MjNhaP1 conclusions • Recombinant reconstituted Mj antiporter is active at pH6 • Inactive at pH7.5 • Drop in pH from 8 to acidic causes ~2Å movement of helix bundle, in addition to reorientation of helices within bundle • Major differences to NhaA in projection structure • Different mechanism of pH activation and ion translocation ?

  20. MjNhaP1: K.R.Vinothkumar Sander Smits Panchali Goswami Özkan Yildiz NhaA: Matthias AppelCarola Hunte K.R.Vinothkumar Elena Scrapanti Karen Williams Etana Padan Hartmut Michel (x-ray structure)

  21. Max Planck Institute of Biophysics Frankfurt

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