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.
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References
Baker, J. L. et al. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235 (2012).
Stockbridge, R. B. et al. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc. Natl. Acad. Sci. USA 109, 15289–15294 (2012).
DeAngeli, A. et al. The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442, 939–942 (2006).
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).
Brammer, A. E., Stockbridge, R. B. & Miller, C. F–/Cl– selectivity in CLCF-type F–/H+ antiporters. J. Gen. Physiol. 144, 129–136 (2014).
Accardi, A. Structure and gating of CLC channels and exchangers. J. Physiol. (Lond.) 593, 4129–4138 (2015).
Dutzler, R., Campbell, E. B. & MacKinnon, R. Gating the selectivity filter in ClC chloride channels. Science 300, 108–112 (2003).
Miller, C. ClC chloride channels viewed through a transporter lens. Nature 440, 484–489 (2006).
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).
Accardi, A. et al. Separate ion pathways in a Cl–/H+ exchanger. J. Gen. Physiol. 126, 563–570 (2005).
Lim, H. H. & Miller, C. Intracellular proton-transfer mutants in a CLC Cl–/H+ exchanger. J. Gen. Physiol. 133, 131–138 (2009).
Accardi, A. & Miller, C. Secondary active transport mediated by a prokaryotic homologue of ClC Cl– channels. Nature 427, 803–807 (2004).
Picollo, A. & Pusch, M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436, 420–423 (2005).
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).
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).
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).
Khantwal, C. M. et al. Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter. eLife 5, e11189 (2016).
Walden, M. et al. Uncoupling and turnover in a Cl–/H+ exchange transporter. J. Gen. Physiol. 129, 317–329 (2007).
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).
Last, N. B., Sun, S., Pham, M. C. & Miller, C. Molecular determinants of permeation in a fluoride-specific ion channel. eLife 6, e31259 (2017).
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).
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).
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).
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).
Phillips, S. et al. Surprises from an unusual CLC homolog. Biophys. J. 103, L44–L46 (2012).
Zhang, Y. & Voth, G. A. The coupled proton transport in the ClC-ec1 Cl–/H+ antiporter. Biophys. J. 101, L47–L49 (2011).
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).
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).
Accardi, A. & Picollo, A. CLC channels and transporters: proteins with borderline personalities. Biochim. Biophys. Acta 1798, 1457–1464 (2010).
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).
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).
Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D. Biol. Crystallogr. 67, 282–292 (2011).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr. 67, 235–242 (2011).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008).
Stamm, M., Staritzbichler, R., Khafizov, K. & Forrest, L. R. AlignMe: a membrane protein sequence alignment web server. Nucleic Acids Res 42, W246–W251 (2014).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).
Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).
Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008).
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).
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All authors performed experiments, N.B.L., R.B.S., and C.M. wrote the paper.
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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.
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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
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DOI: https://doi.org/10.1038/s41594-018-0082-0
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