Article | Published:

Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters

Nature Structural & Molecular Biology volume 23, pages 248255 (2016) | Download Citation

Abstract

To fully understand the transport mechanism of Na+/H+ exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog GltPh. Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na+/H+ antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na+/H+ antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

References

  1. 1.

    , & Evolutionary origins of eukaryotic sodium/proton exchangers. Am. J. Physiol. Cell Physiol. 288, C223–C239 (2005).

  2. 2.

    & Traditional and emerging roles for the SLC9 Na+/H+ exchangers. Pflugers Arch. 466, 61–76 (2014).

  3. 3.

    , , & Implications of sodium hydrogen exchangers in various brain diseases. J. Basic Clin. Physiol. Pharmacol. 26, 417–426 (2015).

  4. 4.

    et al. Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature 435, 1197–1202 (2005).

  5. 5.

    et al. Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights. J. Gen. Physiol. 144, 529–544 (2014).

  6. 6.

    et al. A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573–577 (2013).

  7. 7.

    , , , & Revealing the ligand binding site of NhaA Na+/H+ antiporter and its pH dependence. J. Biol. Chem. 287, 38150–38157 (2012).

  8. 8.

    , & Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. eLife 3, e03579 (2014).

  9. 9.

    et al. Mechanism of Na+/H+ antiporting. Science 317, 799–803 (2007).

  10. 10.

    , & Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880–885 (2009).

  11. 11.

    , , , & Structure and transport mechanism of the sodium/proton antiporter MjNhaP1. eLife 3, e03583 (2014).

  12. 12.

    , , & Functional characterization of a NapA Na+/H+ antiporter from Thermus thermophilus. FEBS Lett. 581, 572–578 (2007).

  13. 13.

    et al. NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter. Biochim. Biophys. Acta 1658, 2–13 (2004).

  14. 14.

    & Conformational changes in NhaA Na+/H+ antiporter. Mol. Membr. Biol. 30, 90–100 (2013).

  15. 15.

    , & The unwound portion dividing helix IV of NhaA undergoes a conformational change at physiological pH and lines the cation passage. Biochemistry 51, 9560–9569 (2012).

  16. 16.

    et al. Transport domain unlocking sets the uptake rate of an aspartate transporter. Nature 518, 68–73 (2015).

  17. 17.

    & Proton/sodium ion antiport in Escherichia coli. Biochem. J. 144, 87–90 (1974).

  18. 18.

    , & Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli. J. Biol. Chem. 266, 11289–11294 (1991).

  19. 19.

    , , & Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: an electrophysiological study. J. Biol. Chem. 286, 23570–23581 (2011).

  20. 20.

    , , & Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers. J. Biol. Chem. 289, 13168–13176 (2014).

  21. 21.

    , , , & Species differences in bacterial NhaA Na+/H+ exchangers. FEBS Lett. 588, 3111–3116 (2014).

  22. 22.

    et al. Crystal structure of a phosphorylation-coupled saccharide transporter. Nature 473, 50–54 (2011).

  23. 23.

    et al. Crystal structure of a phosphorylation-coupled vitamin C transporter. Nat. Struct. Mol. Biol. 22, 238–241 (2015).

  24. 24.

    , & Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å. Nature 483, 489–493 (2012).

  25. 25.

    et al. Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology. Nat. Commun. 6, 6874 (2015).

  26. 26.

    et al. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps. Cell Reports 11, 61–70 (2015).

  27. 27.

    , , & Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT. Nature 478, 408–411 (2011).

  28. 28.

    et al. Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505, 569–573 (2014).

  29. 29.

    , & pH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii. EMBO J. 24, 2720–2729 (2005).

  30. 30.

    & pH- and sodium-induced changes in a sodium/proton antiporter. eLife 3, e01412 (2014).

  31. 31.

    & Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA. Proc. Natl. Acad. Sci. USA 108, 15769–15774 (2011).

  32. 32.

    , , , & Conformations of NhaA, the Na+/H+ exchanger from Escherichia coli, in the pH-activated and ion-translocating states. J. Mol. Biol. 388, 659–672 (2009).

  33. 33.

    , , & Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811–818 (2004).

  34. 34.

    et al. Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. USA 105, 14371–14376 (2008).

  35. 35.

    et al. MemStar: a one-shot Escherichia coli-based approach for high-level bacterial membrane protein production. FEBS Lett. 588, 3761–3769 (2014).

  36. 36.

    , & Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase. Biochim. Biophys. Acta 1706, 110–116 (2005).

  37. 37.

    , & Functional asymmetry of the F(0) motor in bacterial ATP synthases. Mol. Microbiol. 72, 479–490 (2009).

  38. 38.

    XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  39. 39.

    & How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

  40. 40.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  41. 41.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  42. 42.

    , , & MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 32, W615–W619 (2004).

  43. 43.

    & Electron-density map interpretation. Methods Enzymol. 277, 173–208 (1997).

  44. 44.

    & A super position. Joint CCP4 ESF-EACBM Newsl. Protein Crystallogr. 31, 9–14 (1994).

  45. 45.

    et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299–W302 (2005).

  46. 46.

    et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013).

  47. 47.

    et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

  48. 48.

    , & Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004).

  49. 49.

    et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ(1) and χ(2) dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012).

  50. 50.

    et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

  51. 51.

    et al. Coarse-grained MD simulations of membrane protein-bilayer self-assembly. Structure 16, 621–630 (2008).

  52. 52.

    & From coarse grained to atomistic: a serial multiscale approach to membrane protein simulations. J. Chem. Theory Comput. 7, 1157–1166 (2011).

  53. 53.

    , , & Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J. Chem. Theory Comput. 7, 2284–2295 (2011).

  54. 54.

    , & Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007).

  55. 55.

    & Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

  56. 56.

    et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8592 (1995).

  57. 57.

    , & Molecular dynamics simulations of phosphatidylcholine membranes: a comparative force field study. J. Chem. Theory Comput. 8, 4593–4609 (2012).

  58. 58.

    P-LINCS:a parallel linear constraint solver for molecular simulation. J. Chem. Theory Comput. 4, 116–122 (2008).

  59. 59.

    & SETTLE: an analytical version of the SHAKE and RATTLE algorithms for rigid water models. J. Comput. Chem. 13, 952–962 (1992).

  60. 60.

    , , & MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. J. Comput. Chem. 32, 2319–2327 (2011).

  61. 61.

    , , & OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006).

  62. 62.

    , & VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996).

  63. 63.

    , & Bendix: intuitive helix geometry analysis and abstraction. Bioinformatics 28, 2193–2194 (2012).

Download references

Acknowledgements

We are grateful to G. Verdon for discussions and comments. Data were collected at Diamond Light Source with excellent assistance from beamline scientists. This work was supported by the Swedish Research Council (D.D.) and the Knut and Alice Wallenberg Foundation (D.D.). The authors are grateful for the use of the Membrane Protein Laboratory supported by the Wellcome Trust UK (grant 062164/Z/00/Z) at the Diamond Light Source Limited and the Centre for Biomembrane Research supported by the Swedish Foundation for Strategic Research. Computer simulations were partially run on the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by US National Science Foundation grant OCI-1053575 (allocation TG-MCB130177 to O.B.). M.C. was supported as a Wenner-Gren postdoctoral fellow, and D.D. is supported as a European Molecular Biology Organization (EMBO) Young Investigator.

Author information

Author notes

    • Mathieu Coincon
    •  & Povilas Uzdavinys

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

    • Mathieu Coincon
    • , Povilas Uzdavinys
    • , Emmanuel Nji
    • , Iven Winkelmann
    • , Saba Abdul-Hussein
    •  & David Drew
  2. Department of Physics, Arizona State University, Tempe, Arizona, USA.

    • David L Dotson
    •  & Oliver Beckstein
  3. Center for Biological Physics, Arizona State University, Tempe, Arizona, USA.

    • David L Dotson
    •  & Oliver Beckstein
  4. School of Life Sciences, University of Warwick, Coventry, UK.

    • Alexander D Cameron

Authors

  1. Search for Mathieu Coincon in:

  2. Search for Povilas Uzdavinys in:

  3. Search for Emmanuel Nji in:

  4. Search for David L Dotson in:

  5. Search for Iven Winkelmann in:

  6. Search for Saba Abdul-Hussein in:

  7. Search for Alexander D Cameron in:

  8. Search for Oliver Beckstein in:

  9. Search for David Drew in:

Contributions

D.D. designed the project. Cloning, expression screening, protein purification and crystallization of inward-facing NapA were carried out by M.C. and P.U. LCP crystallization of outward-facing NapA was carried out by E.N. Data collection and structural determination were carried out by M.C. and D.D. with assistance from E.N. and A.D.C. Experiments for functional analysis were carried out by P.U., I.W., S.A.-H. and M.C. MD simulations were carried out by D.L.D. and O.B. D.D. wrote the manuscript with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David Drew.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1 and 2

  2. 2.

    Supplementary Data Set 1

    Uncropped SDS-gels used to assess disulfide-bond formation of NapA cysteine mutants

Videos

  1. 1.

    The elevator alternating-access mechanism of the Na+ /H+ antiporter NapA

    Video showing the morph between the outwardfacing and inward-facing NapA crystal structures after superposition against the dimer domains. The colouring as in Fig. 1a except for half-helices TM11b and TM4b shown in orange and magenta, respectively. The strictly conserved ion-binding aspartate D157 is shown in stick form. View is from the side.

  2. 2.

    The elevator alternating-access mechanism of the Na+ /H+ antiporter NapA

    As in Video 1 viewed from the extracellular surface

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3164

Further reading