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Crystal structures of a double-barrelled fluoride ion channel

Nature volume 525, pages 548–551 (2015)Cite this article

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Abstract

To contend with hazards posed by environmental fluoride, microorganisms export this anion through F−-specific ion channels of the Fluc family1,2,3,4. Since the recent discovery of Fluc channels, numerous idiosyncratic features of these proteins have been unearthed, including strong selectivity for F− over Cl− and dual-topology dimeric assembly5,6. To understand the chemical basis for F− permeation and how the antiparallel subunits convene to form a F−-selective pore, here we solve the crystal structures of two bacterial Fluc homologues in complex with three different monobody inhibitors, with and without F− present, to a maximum resolution of 2.1 Å. The structures reveal a surprising ‘double-barrelled’ channel architecture in which two F− ion pathways span the membrane, and the dual-topology arrangement includes a centrally coordinated cation, most likely Na+. F− selectivity is proposed to arise from the very narrow pores and an unusual anion coordination that exploits the quadrupolar edges of conserved phenylalanine rings.

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Figure 1: Bpe–S7 structure.
Figure 2: Bpe–L2 structure.
Figure 3: Identification of F− ions.
Figure 4: Model of F− movement by Fluc ‘channsporter’ mechanism.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinate files have been uploaded to the Protein Data Bank (PDB) with accession codes 5A40, 5A41 and 5A43 for the Bpe–S7, Bpe–L2 and Ec2–S9 complexes, respectively.

References

  1. Baker, J. L. et al. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235 (2012)

    Article  ADS  CAS  Google Scholar 

  2. Li, S. et al. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins. Proc. Natl Acad. Sci. USA 110, 19018–19023 (2013)

    Article  ADS  CAS  Google Scholar 

  3. Ji, C., Stockbridge, R. B. & Miller, C. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J. Gen. Physiol. 144, 257–261 (2014)

    Article  CAS  Google Scholar 

  4. Smith, K. D. et al. Yeast FEX1 is a constitutively expressived fluoride channel in with functional asymmetry of its two homologous domains. J. Biol. Chem. 290, 19874–19887 (2015)

    Article  CAS  Google Scholar 

  5. Stockbridge, R. B., Robertson, J. L., Kolmakova-Partensky, L. & Miller, C. A family of fluoride-specific ion channels with dual-topology architecture. eLife 2, e01084. (2013)

    Article  Google Scholar 

  6. Stockbridge, R. B., Koide, A., Miller, C. & Koide, S. Proof of dual-topology architecture of Fluc F− channels with monobody blockers. Nature Commun. 5, 5120 (2014)

    Article  ADS  CAS  Google Scholar 

  7. Adamek, E., Pawlowska-Goral, K. & Bober, K. In vitro and in vivo effects of fluoride ions on enzyme activity. Ann. Acad. Med. Stetin. 51, 69–85 (2005)

    CAS  PubMed  Google Scholar 

  8. Stockbridge, R. B. et al. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc. Natl Acad. Sci. USA 109, 15289–15294 (2012)

    Article  ADS  CAS  Google Scholar 

  9. Ubarretxena-Belandia, I., Baldwin, J. M., Schuldiner, S. & Tate, C. G. Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer. EMBO J. 22, 6175–6181 (2003)

    Article  CAS  Google Scholar 

  10. Schuldiner, S. EmrE, a model for studying evolution and mechanism of ion-coupled transporters. Biochim. Biophys. Acta 1794, 748–762 (2009)

    Article  CAS  Google Scholar 

  11. Morrison, E. A. et al. Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481, 45–50 (2011)

    Article  ADS  Google Scholar 

  12. Miller, C. Open-state substructure of single chloride channels from Torpedo electroplax. Philos. Trans. R. Soc. Lond. B 299, 401–411 (1982)

    Article  ADS  CAS  Google Scholar 

  13. Rapp, M., Granseth, E., Seppala, S. & von Heijne, G. Identification and evolution of dual-topology membrane proteins. Nature Struct. Mol. Biol. 13, 112–116 (2006)

    Article  CAS  Google Scholar 

  14. Koide, A., Wojcik, J., Gilbreth, R. N., Hoey, R. J. & Koide, S. Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold. J. Mol. Biol. 415, 393–405 (2012)

    Article  CAS  Google Scholar 

  15. Turman, D. L., Nathanson, J. T., Stockbridge, R. B., Street, T. O. & Miller, C. Two-sided block of a dual-topology F– channel. Proc. Natl Acad. Sci. USA 112, 5697–5701 (2015)

    Article  ADS  CAS  Google Scholar 

  16. Harding, M. M. Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr. D 58, 872–874 (2002)

    Article  Google Scholar 

  17. Varma, S., Sabo, D. & Rempe, S. B. K+/Na+ selectivity in K channels and valinomycin: over-coordination versus cavity-size constraints. J. Mol. Biol. 376, 13–22 (2008)

    Article  CAS  Google Scholar 

  18. Lev, B. R. B. & Noskov, S. Y. in Encyclopedia of Metalloproteins (eds Kretsinger, R. H. et al.) 2112–2118 (Springer, 2013)

    Book  Google Scholar 

  19. Philip, V. et al. A survey of aspartate-phenylalanine and glutamate-phenylalanine interactions in the protein data bank: searching for anion-π pairs. Biochemistry 50, 2939–2950 (2011)

    Article  CAS  Google Scholar 

  20. Allen, F. H. The Cambridge Structural Database: a quarter of a million crystal structures and rising. Acta Crystallogr. B 58, 380–388 (2002)

    Article  Google Scholar 

  21. Kane Dickson, V., Pedi, L. & Long, S. B. Structure and insights into the function of a Ca2+-activated Cl− channel. Nature 516, 213–218 (2014)

    Article  ADS  Google Scholar 

  22. Walden, M. et al. Uncoupling and turnover in a Cl−/H+ exchange transporter. J. Gen. Physiol. 129, 317–329 (2007)

    Article  CAS  Google Scholar 

  23. Brammer, A. E., Stockbridge, R. B. & Miller, C. F. F−/Cl− selectivity in CLCF-type F−/H+ antiporters. J. Gen. Physiol. 144, 129–136 (2014)

    Article  CAS  Google Scholar 

  24. Weinreich, F. & Jentsch, T. J. Pores formed by single subunits in mixed dimers of different CLC chloride channels. J. Biol. Chem. 276, 2347–2353 (2001)

    Article  CAS  Google Scholar 

  25. Hille, B. & Schwarz, W. Potassium channels as multi-ion single-file pores. J. Gen. Physiol. 72, 409–442 (1978)

    Article  CAS  Google Scholar 

  26. Morais-Cabral, J. H., Zhou, Y. & MacKinnon, R. Energetic optimization of ion conduction rate by the K+ selectivity filter. Nature 414, 37–42 (2001)

    Article  ADS  CAS  Google Scholar 

  27. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

    Article  CAS  Google Scholar 

  28. Winter, G., Lobley, C. M. & Prince, S. M. Decision making in xia2. Acta Crystallogr. D 69, 1260–1273 (2013)

    Article  CAS  Google Scholar 

  29. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  Google Scholar 

  30. Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011)

    Article  CAS  Google Scholar 

  31. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    Article  ADS  CAS  Google Scholar 

  32. Bricogne, G. et al. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

    Article  CAS  Google Scholar 

  33. Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

    Article  CAS  Google Scholar 

  34. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  35. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  36. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

    Article  CAS  Google Scholar 

  37. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

    Article  CAS  Google Scholar 

  38. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    Article  CAS  Google Scholar 

  39. Chen, V. B. et al. Molprobity: all-atom structure validataion for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66 486–501 http://dx.doi.org/10.1107/S0907444910007493 (2010)

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to J. Parker for critically reading the manuscript; to D. Turman, A. Lajoie, M. Pham, and K. Piasta for pilot experiments; to S. Strobel for sharing results of unpublished experiments; and to B. Foxman for help with the Cambridge Structural Database. This work was supported in part by a Wellcome Trust Investigator Award 102890/Z/13/Z and National Institutes of Health (NIH) grants RO1-GM107023 and U54-GM087519. R.B.S. was supported by NIH grant K99-GM-111767. C.M. is grateful to F. Ashcroft and Lincoln College, Oxford, for hosting a Newton-Abraham Visiting Professorship.

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Authors and Affiliations

  1. Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, 02454, Massachusetts, USA

    Randy B. Stockbridge, Ludmila Kolmakova-Partensky, Tania Shane & Christopher Miller

  2. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, 60637, Illinois, USA

    Akiko Koide & Shohei Koide

  3. Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, OX1 3QU, UK

    Christopher Miller

  4. Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK

    Simon Newstead

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Contributions

R.B.S., S.N. and C.M. performed experiments, analysed the data and wrote the paper. L.K.-P. and T.S. performed experiments. S.K. and A.K. provided monobody clones and wrote the paper.

Corresponding author

Correspondence to Simon Newstead.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Crystal lattices for the Bpe–S7, Bpe–L2 and Ec2–S9 crystal structures.

The asymmetric unit is shown in green and red (channel and monobody, respectively), and symmetry mates are shown in black and blue.

Extended Data Figure 2 Bpe–L2 complex.

Left, cartoon schematic of Bpe crystal structure, coloured as in Fig. 1b. The variable regions of monobody L2 are coloured cyan. Mesh-rendering is shown for the lower monobody. Right, single-channel recording of Bpe in the presence of 200 nM L2. Zero-current level is indicated by the dashed line.

Extended Data Figure 3 Stereo images of Bpe–L2.

a–d, Stereo images corresponding to the structures shown in Fig. 2a–d.

Extended Data Figure 4 Single channel trace of Bpe in Na+-free recording solution, with addition of 200 nM blocking monobody L3.

Channels were recorded in the presence of 300 mM N-methyl-glucamine-fluoride, from which all small cations were rigorously excluded. The zero-current level is indicated by the dashed line.

Extended Data Figure 5 Experimental electron density for the Ec2–S9 crystal structure.

Left, cartoon schematic of Ec2 with S9 monobodies bound, coloured as in Bpe in Fig. 1b. Variable sequences of the monobodies (cyan) with ribbon or mesh representation. Right, cartoon view of TM4 from Ec2, with the solvent-flattened electron density map calculated from SHARP contoured at 1.8σ (blue), and anomalous difference density from seleno-l-methionine contoured at 5σ (magenta).

Extended Data Figure 6 Liposome flux assays of Bpe variants.

Top three panels: F− transport from liposomes by Bpe mutants F82I, F85I, and N43D, in the presence and absence of 6 μM blocking monobody. F− efflux from proteoliposomes (0.2 μg protein per mg lipid for Phe mutants; 1 μg protein per mg lipid for N43D) was monitored with a F− electrode and normalized against total trapped F−. Bottom panel: F− dump by F85I measured at pH 7 and pH 9. Rates are summarized in Extended Data Table 2.

Extended Data Figure 7 Sequence alignment of eukaryotic N- and C-terminal Fluc domain sequences, with bacterial homodimer sequences below.

Highly conserved residues are shaded in grey. For the eukaryotic sequences, residues expected to line ‘pore 2’, the pore mostly encompassed by the C-terminal domain, are coloured red.

Extended Data Table 1 Data collection, phasing and refinement statistics
Full size table
Extended Data Table 2 F− turnover rate for Bpe mutants
Full size table

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Stockbridge, R., Kolmakova-Partensky, L., Shane, T. et al. Crystal structures of a double-barrelled fluoride ion channel. Nature 525, 548–551 (2015). https://doi.org/10.1038/nature14981

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