Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A CLC-type F-/H+ antiporter in ion-swapped conformations

Abstract

Fluoride/proton antiporters of the CLCF family combat F toxicity in bacteria by exporting this halide from the cytoplasm. These transporters belong to the widespread CLC superfamily but display transport properties different from those of the well-studied Cl/H+ antiporters. Here, we report a structural and functional investigation of these F-transport proteins. Crystal structures of a CLCF homolog from Enterococcus casseliflavus are captured in two conformations with simultaneous accessibility of F and H+ ions via separate pathways on opposite sides of the membrane. Manipulation of a key glutamate residue critical for H+ and F transport reverses the anion selectivity of transport; replacement of the glutamate with glutamine or alanine completely inhibits F and H+ transport while allowing for rapid uncoupled flux of Cl. The structural and functional results lead to a ‘windmill’ model of CLC antiport wherein F and H+ simultaneously move through separate ion-specific pathways that switch sidedness during the transport cycle.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Ion transport for Eca and ion-coupling mutants.
Fig. 2: Structure of CLCF-Eca.
Fig. 3: Ion-coupling region of Eca.
Fig. 4: F block of Cl flux in Glug mutants.
Fig. 5: Ion accessibility of WT Eca and V319G mutant.
Fig. 6: F/H+ antiport model.

Similar content being viewed by others

References

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  3. DeAngeli, A. et al. The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442, 939–942 (2006).

    Article  CAS  Google Scholar 

  4. Picollo, A., Malvezzi, M., Houtman, J. C. & Accardi, A. Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters. Nat. Struct. Mol. Biol. 16, 1294–1301 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Accardi, A. Structure and gating of CLC channels and exchangers. J. Physiol. (Lond.) 593, 4129–4138 (2015).

    Article  CAS  Google Scholar 

  7. Dutzler, R., Campbell, E. B. & MacKinnon, R. Gating the selectivity filter in ClC chloride channels. Science 300, 108–112 (2003).

    Article  PubMed  CAS  Google Scholar 

  8. Miller, C. ClC chloride channels viewed through a transporter lens. Nature 440, 484–489 (2006).

    Article  PubMed  CAS  Google Scholar 

  9. Vien, M., Basilio, D., Leisle, L. & Accardi, A. Probing the conformation of a conserved glutamic acid within the Cl pathway of a CLC H+/Cl exchanger. J. Gen. Physiol. 149, 523–529 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Accardi, A. et al. Separate ion pathways in a Cl/H+ exchanger. J. Gen. Physiol. 126, 563–570 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Lim, H. H. & Miller, C. Intracellular proton-transfer mutants in a CLC Cl/H+ exchanger. J. Gen. Physiol. 133, 131–138 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Accardi, A. & Miller, C. Secondary active transport mediated by a prokaryotic homologue of ClC Cl channels. Nature 427, 803–807 (2004).

    Article  PubMed  CAS  Google Scholar 

  13. Picollo, A. & Pusch, M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436, 420–423 (2005).

    Article  PubMed  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  PubMed  CAS  Google Scholar 

  15. Feng, L., Campbell, E. B., Hsiung, Y. & MacKinnon, R. Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle. Science 330, 635–641 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Basilio, D., Noack, K., Picollo, A. & Accardi, A. Conformational changes required for H+/Cl exchange mediated by a CLC transporter. Nat. Struct. Mol. Biol. 21, 456–463 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Khantwal, C. M. et al. Revealing an outward-facing open conformational state in a CLC Cl/H+ exchange transporter. eLife 5, e11189 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Jayaram, H., Accardi, A., Wu, F., Williams, C. & Miller, C. Ion permeation through a Cl-selective channel designed from a CLC Cl/H+ exchanger. Proc. Natl. Acad. Sci. USA 105, 11194–11199 (2008).

    Article  PubMed  Google Scholar 

  20. Last, N. B., Sun, S., Pham, M. C. & Miller, C. Molecular determinants of permeation in a fluoride-specific ion channel. eLife 6, e31259 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  21. 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  PubMed  PubMed Central  Google Scholar 

  22. Last, N. B., Kolmakova-Partensky, L., Shane, T. & Miller, C. Mechanistic signs of double-barreled structure in a fluoride ion channel. eLife 5, 5.e18767 (2016).

    Article  CAS  Google Scholar 

  23. Picollo, A., Xu, Y., Johner, N., Bernèche, S. & Accardi, A. Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H+/Cl exchanger. Nat. Struct. Mol. Biol. 19, 525–531 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Lim, H. H., Stockbridge, R. B. & Miller, C. Fluoride-dependent interruption of the transport cycle of a CLC Cl–/H+ antiporter. Nat. Chem. Biol. 9, 721–725 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Phillips, S. et al. Surprises from an unusual CLC homolog. Biophys. J. 103, L44–L46 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Zhang, Y. & Voth, G. A. The coupled proton transport in the ClC-ec1 Cl/H+ antiporter. Biophys. J. 101, L47–L49 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Jiang, T., Han, W., Maduke, M. & Tajkhorshid, E. Molecular basis for differential anion binding and proton coupling in the Cl/H+ exchanger ClC-ec1. J. Am. Chem. Soc. 138, 3066–3075 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Miller, C. & Nguitragool, W. A provisional transport mechanism for a chloride channel-type Cl/H+ exchanger. Philos. Trans. R. Soc. Lond. B 364, 175–180 (2009).

    Article  CAS  Google Scholar 

  29. Accardi, A. & Picollo, A. CLC channels and transporters: proteins with borderline personalities. Biochim. Biophys. Acta 1798, 1457–1464 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Lee, S., Swanson, J. M. & Voth, G. A. Multiscale simulations reveal key aspects of the proton transport mechanism in the ClC-ec1 antiporter. Biophys. J. 110, 1334–1345 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Skerra, A. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151, 131–135 (1994).

    Article  PubMed  CAS  Google Scholar 

  32. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008).

    Article  CAS  Google Scholar 

  37. Stamm, M., Staritzbichler, R., Khafizov, K. & Forrest, L. R. AlignMe: a membrane protein sequence alignment web server. Nucleic Acids Res 42, W246–W251 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003).

    Article  PubMed  CAS  Google Scholar 

  40. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank members of the laboratory of C.M. for advice and criticism, and we acknowledge with ambiguous gratitude frequent and interminable discussions with A. Accardi. We are unambiguously grateful to the beamline scientists for their expert help with resources at the Advanced Light Source, a DOE User Facility, contract DE-AC02-05CH11231. This project was supported in part by NIH grants R01-GM107023 (C.M.), U54-GM087519 (S.K.), and S10OD021832 (for ALS beamline 5.0.1).

Author information

Authors and Affiliations

Authors

Contributions

All authors performed experiments, N.B.L., R.B.S., and C.M. wrote the paper.

Corresponding author

Correspondence to Christopher Miller.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Eca electron density in ion-binding region.

a, c. F- ion omit mFo-DFc maps (green mesh) in WT Eca contoured to 4 σ (a) or E118Q contoured at 3.5 σ (c). Glug sidechain (orange stick) is also indicated in all panels b, d. Stereo images of 2mFo-DFc density contoured to 1.2 σ (grey) in WT (b) or E118Q (d).

Supplementary Figure 2 Eca alignment with CLC-ec1.

a. Overlay of WT Eca subunit (cyan) with Cl-/H+ antiporter CLC-ec1 (grey, PDB# 1OTS). b. Alignment of ion-coupling regions of WT Eca with CLC-ec1 E148Q (PDB# 1OTU), with mutated Glug sidechain in Up-position. Positions of Eca F- (magenta) and CLC-ec1 Cl- (yellow) are also indicated.

Supplementary Figure 3 F binding affinity from ITC experiments.

F- titrations (from 3 repeats each) for WT (left) and V319G (right). Solid lines represent KD = 0.20 mM, 1.0 mM, respectively.

Supplementary Figure 4 V319G electron density and sequence alignment.

a. F- ion difference density. mFo-DFc map refined in the absence of F- (green mesh) contoured to 4 σ. b. Stereo view of 2mFo-DFc density contoured to 1.2 σ (grey) in the region surrounding Glug (orange stick). c. Alignment of ion-coupling regions of V319G Eca with the CLC Cl-/H+ antiporter from C. merolae (brown ribbon, yellow Cl-, PDB 3ORG).

Supplementary Figure 5 Rotameric gymnastics of Glug in the Eca antiport cycle.

Proposed pathway of Glug from Up to Down conformations in Step 1 of transport cycle of Fig. 6. Rotamers shown allow F-ex to remain bound during the transition. This motion would likely require movement of F154, shown at right. Y396 is also shown below.

Supplementary Figure 6 Monobody crystallization chaperone sequence.

Complete sequence of the monobody “X1” used for crystallization. Residues in red are removed upon TEV cleavage, leaving the black residues as the protein used to complex Eca. Residue numbering in the structures is based on the cleaved protein. Penultimate residue (underlined) is an arginine in the V319G structure.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Last, N.B., Stockbridge, R.B., Wilson, A.E. et al. A CLC-type F-/H+ antiporter in ion-swapped conformations. Nat Struct Mol Biol 25, 601–606 (2018). https://doi.org/10.1038/s41594-018-0082-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-018-0082-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing