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Cited by 120Ion channel regulation and functionIon Transport and Channel RegulationEnzyme Structure and Function

The DOI matches its Crossref record; 42 of the 42 references it cites were found in a registry or an index; none of the 47 sources its claims cite is checked for support yet.

Crystal structures of a double-barrelled fluoride ion channel

Open access
Stockbridge, Randy B., Kolmakova-Partensky, Ludmila, Shane, Tania, Koide, Akiko, Koide, Shohei, Miller, Christopher, Newstead, Simon·2015·Nature
DOI 10.1038/nature14981PMC PMC4876929
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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 family. 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 assembly. 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.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Crystal structures of bacterial Fluc homologues Bpe and Ec2 in complex with monobody inhibitors reveal a “double-barrelled” antiparallel homodimer with two narrow F⁻ pathways and a centrally coordinated Na⁺. Mutational and flux assays support key roles for conserved phenylalanines and N43 in F⁻ permeation and selectivity.

Key findings

  • Structures solved to a maximum resolution of 2.1 Å show an antiparallel homodimer with a novel fold, a ~1700 Ų membrane-embedded dimer interface, and a central plug containing a Na⁺ coordinated by backbone carbonyls from the conserved TM3 break (G77, T80).
  • The Bpe-L2 structure contains four densities provisionally assigned as F⁻ ions in peripheral crevices, suggesting two antiparallel pores rather than a single central pore; Ec2-S9 shows equivalent densities at the F1 positions, with Cα rmsd 0.6 Å between Bpe and Ec2 folds.
  • F⁻ selectivity is proposed to arise from very narrow pores and unusual anion coordination involving conserved phenylalanine rings (F82, F85) in side-on/quadrupolar orientation, plus H-bond donors including N43, S108, and S112.
  • Anion-dump assays: wild-type Bpe F⁻ efflux is ~3×10⁵ s⁻¹; F85I is 1950 ± 200 s⁻¹ and F82I is 200 ± 12 s⁻¹; N43D efflux is 4330 ± 440 s⁻¹ at pH 6.5, 1860 ± 210 s⁻¹ at pH 7, and undetectable at pH 9.

Methods

  • Crystallized Bpe with monobodies S7 and L2, and Ec2 with monobody S9, using detergent micelles or lipidic cubic phase; Bpe-S7 phases were determined by Hg-SAD and improved with Hg/Se-SIRAS, Bpe-L2 by molecular replacement, and Ec2-S9 by Se-SAD.
  • Functional measurements used single-channel recording in planar lipid bilayers and electrochemical anion-efflux assays from proteoliposomes loaded with KF or KCl, including Bpe mutants F82I, F85I, and N43D.
  • Structural analysis included model building/refinement, sequence alignments of eukaryotic and bacterial Fluc domains, and comparison of conserved pore-lining residues.

Limitations

  • The authors state that the low-resolution Bpe-S7 structure lacks F⁻ and does not reveal the ion-permeation pathway; they also state that the evidence for assigning the four densities as F⁻ is not sufficiently strong to accept the double-barrelled picture as firmly established.
  • The authors note that monobodies might force a conformation in which the two pores are less open than in the fully conducting state, that Ec2 densities corresponding to F2 cannot be unambiguously assigned, and that the energetic contributions of phenylalanine edge-on coordination have not been established.
  • Evident from the text: the double-barrelled architecture is inferred from crystallographic densities and functional mutagenesis rather than a direct open-state structure, and N43S/N43A mutants were biochemically intractable, limiting analysis of that position.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/nature14981

Cites

42 of 42 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 47 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The dual-topology arrangement includes a centrally coordinated cation, most likely Na+.

    “the dual-topology arrangement includes a centrally coordinated cation, most likely Na+.”
    Quote in the paperNot a substantial claim
  • Findingown data

    The authors solved 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.

    “we solved 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.”
    Quote in the paperSubstantial claim
  • Findingown data

    The structures reveal a double-barrelled channel architecture in which two F- ion pathways span the membrane.

    “The structures reveal a surprising “double-barrelled” channel architecture in which two F- ion pathways span the membrane”
    Quote in the paperSubstantial claim
  • Findingown data

    Four electron densities are located in crevices between TM2, TM3b, and TM4 near the channel's periphery, distant from the vestibules and the central plug.

    “a set of four electron densities located in crevices between TM2, TM3b, and TM4 near the channel’s periphery, distant from the vestibules and the central plug”
    Quote in the paperSubstantial claim
  • Findingown data

    A strongly conserved asparagine (N43) in TM2 and two conserved serines (S108, S112) in TM4 are prominent in the surround.

    “Prominent among these are a strongly conserved asparagine (N43) in TM2 and two conserved serines (S108, S112) in TM4.”
    Quote in the paperSubstantial claim
  • The quote only partly states this claim. 1
  • It may state two separate claims as one. 35
  • Probably not a substantial claim. 26

  • The quote only partly states this claim.

    Findingown data

    Diffraction to 3.6 Å Bragg spacing was obtained with monobody Mb(Bpe-S7).

    “however diffraction to 3.6 Å Bragg spacing was obtained with monobody Mb(Bpe-S7)6, hereafter denoted S7.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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) source

  • It may state two separate claims as one.

    Findingown data

    Additional densities also appear in Ec2 in the general vicinity as the F2 ions in Bpe, but at lower resolution and without supporting experimental evidence, these densities in Ec2 cannot be unambiguously assigned.

    “Additional densities also appear in Ec2 in the general vicinity as the F2 ions in Bpe, but at this lower resolution and without supporting experimental evidence, these densities in Ec2 cannot be unambiguously assigned.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Most of the channel's aqueous-exposed surface is covered by monobody, consistent with S7 block of Bpe seen in single-channel recordings.

    “Most of the channel’s aqueous-exposed surface is covered by monobody, consistent with S7 block of Bpe seen in single-channel recordings (Fig. 1c).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The channel is hourglass-shaped, with wide vestibules symmetrically opening to the two aqueous solutions separated by a solid plug of protein 10-15 Å thick.

    “The channel is hourglass-shaped, with wide vestibules symmetrically opening to the two aqueous solutions (Fig. 1b) separated by a solid plug of protein 10-15 Å thick.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The Ec2 and Bpe folds are identical (Cα rmsd 0.6 Å), with inferred Na+ density appearing in equivalent locations.

    “The Ec2 and Bpe folds are identical (Cα rmsd 0.6 Å), with inferred Na+ density appearing in equivalent locations.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    These mutations preserve the channel's integrity, since F- efflux is blocked 50-80% by 6μM monobody.

    “These mutations preserve the channel's integrity, since F- efflux is blocked 50-80% by 6µM monobody”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Mutating each of three conserved Bpe residues coordinating the F- densities (F82, F85, N43) did not produce single-channel activity in electrical recordings, but anion-dump experiments reveal dramatic changes in F- permeation.

    “We next examined the functional consequences of mutating each of three conserved Bpe residues coordinating the F- densities: F82, F85, and N43. We did not observe single-channel activity in electrical recordings, but more sensitive “anion-dump” experiments22,23 reveal dramatic changes in F- permeation”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] Walden, M. et al. Uncoupling and turnover in a Cl−/H+ exchange transporter. J. Gen. Physiol. 129, 317–329 (2007) source
    • cites [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) source

  • It may state two separate claims as one.

    Findingown data

    Two strong difference densities lie at precisely the same locations as the F1 ions in Bpe, coordinated identically.

    “Two strong difference densities lie at precisely the same locations as the F1 ions in Bpe, coordinated identically”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingcitation

    All 16 Se plus 4 Hg sites were located using SHELX.

    “All 16 Se plus 4 Hg sites were located using SHELX and this dataset was combined with the initial 3.6 Angstrom Hg derivatized data.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites SHELX

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingown data

    For F85I and F82I, efflux of F- is 2 and 3 orders of magnitude slower, respectively, than for wild type (~3x10^5 s-1).

    “For F85I and F82I, efflux of F- is 2 and 3 orders of magnitude slower, respectively, than for wild type (~3x105 s-1)15”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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) source

  • It may state two separate claims as one.

    Findingown data

    L2 binds in an orientation different from S7's and extends a long loop 8-10 Å into the vestibule, occluding much of the channel's water-exposed surface.

    “L2, though binding in an orientation different from S7's, also extends a long loop 8-10 Å into the vestibule, occluding much of the channel’s water-exposed surface.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    An 8-residue loop on the monobody plunges deeply into each vestibule, contacting the channel mainly via side chains.

    “An 8-residue loop on the monobody plunges deeply into each vestibule, contacting the channel mainly via side chains.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The native Bpe crystals did not show higher resolution diffraction and typically gave diffraction between 3.6 and 3.8 Angstroms.

    “We did not observe higher resolution diffraction in the Native crystals, which typically gave diffraction between 3.6 - 3.8 Angstroms.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    In contrast to pH-independent activity of wild type Bpe and F85I, F- efflux in N43D falls with increasing pH and is extinguished at pH 9.

    “In stark contrast to the pH-independent activity of wild type Bpe and the F85I mutant (Extended Data Fig. 5), F- efflux in N43D falls with increasing pH and is extinguished at pH 9”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Alignments show that "pore-2," where most residues arise from the C-terminal domain, retains the strict sequence conservation typical of the homodimeric bacterial Flucs, while the equivalent residues of "pore-1," mostly in the N-terminal domain, are far less conserved, notably along the TM4 polar track.

    “Alignments (Extended Data Fig. 7) show that “pore-2,” where most residues arise from the C-terminal domain, retains the strict sequence conservation typical of the homodimeric bacterial Flucs, while the equivalent residues of “pore-1,” mostly in the N-terminal domain, are far less conserved, notably along the TM4 polar track.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    These particular residues are only modestly conserved among Fluc channels, but H-bond donors reliably appear here throughout the family.

    “These particular residues are only modestly conserved among Fluc channels, but H-bond donors reliably appear here throughout the family.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Findingown data

    Four Hg sites were located in the Bpe-S7 Hg-derivatised crystals using the single wavelength anomalous dispersion (SAD) method.

    “The four Hg sites were located using the single wavelength anomalous dispersion (SAD) method as implemented in SHELX36 with the positions further refined and initial phases calculated using SHARP37 with solvent flattening in SOLOMON38.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites SHELX36

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites SHARP37

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites SOLOMON38

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingown data

    Bpe is an antiparallel homodimer in which each subunit consists of four transmembrane helices with an overall fold novel among membrane proteins.

    “Bpe is an antiparallel homodimer in which each subunit consists of four transmembrane helices with an overall fold novel among membrane proteins.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The space group of Bpe-S7 crystals was P21212, with two Bpe dimers and four S7 monobodies in the asymmetric unit.

    “The space group was determined to be P21212 with two Bpe dimers and four S7 monobodies in the asymmetric unit (Extended data Figure 1).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingcitation

    The two pathways are not segregated to each subunit as in CLC channels; instead each pore comprises side chains from TM2, TM3b, and TM4 of one subunit plus the TM3-break phenylalanine from the opposing subunit.

    “The two pathways are not segregated to each subunit as in CLC channels24; instead each pore comprises side chains from TM2, TM3b, and TM4 of one subunit plus the TM3-break phenylalanine from the opposing subunit.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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) source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Phases for Ec2-S9 were calculated using Se-SAD with 8 Se sites.

    “Phases were calculated using Se-SAD with 8 Se sites, and processed as above.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    For Bpe-L2 crystals, data were processed and scaled in space group P1.

    “For the Bpe-L2 crystals, data were similarly processed and scaled in spacegroup P1.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    F- turnover by WT exceeds the response time of the electrode.

    “F- turnover by WT exceeds response time of the electrode.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    For Ec2-S9 crystals, data were processed with space group P41.

    “For the Ec2-S9 crystals the data were processed as above with space group P41.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    The electron density maps for Bpe-L2 clearly showed major differences in the selected, variable regions of the monobody.

    “The electron density maps clearly showed major differences in the selected, variable regions of the monobody.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    Refinement of the Bpe-S7 model was improved following anisotropic truncation of the structure factors.

    “No prior phase information was used during the refinement, however refinement was improved following anisotropic truncation of the structure factors.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    The experimental electron density maps for Ec2-S9 were of exceptional quality following phase extension to the highest resolution shell of 2.58 Å.

    “The experimental electron density maps were of exceptional quality following phase extension to the highest resolution shell of 2.58 Å.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Premisecitation

    Fluc proteins are F--specific ion channels of the Fluc family.

    “microorganisms export this anion through F--specific ion channels of the Fluc family1–4.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [1] Baker, J. L. et al. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235 (2012) source
    • cites [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) source
    • cites [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) source
    • cites [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) source

  • It may state two separate claims as one.

    Premisecitation

    Fluc channels exhibit extreme selectivity for F- over Cl- and dual-topology dimeric assembly.

    “including extreme selectivity for F- over Cl- and dual-topology dimeric assembly5–6.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [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) source
    • cites [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) source

  • Probably not a substantial claim.

    Premisecitation

    Fluoride is a xenobiotic inhibitor of essential phosphoryl-transfer enzymes.

    “The fluoride anion, ubiquitous in the aqueous biosphere throughout evolutionary time, is a xenobiotic inhibitor of essential phosphoryl-transfer enzymes7.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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)

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    The channel's extreme F- selectivity may arise from the narrow bore of the permeation pathway, which would exclude Cl- ions while permitting the smaller F- to enter.

    “Third, the channel’s extreme F- selectivity may arise from the narrow bore of the permeation pathway, which would exclude Cl- ions while permitting the smaller F- to enter.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Study design and conductown data

    Ec2-S9 crystals diffracting to 2.6 Å were grown from detergent in the presence of F-, and solved using SAD phasing with selenomethionine-labelled protein to avoid model bias.

    “Ec2-S9 crystals diffracting to 2.6 Å were grown from detergent in the presence of F-, and to avoid model bias a structure was solved using SAD phasing with selenomethionine-labelled protein”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationown data

    A buried Na+ ion occupies a unique position on the twofold axis and is viewed as an intrinsic structural element stabilizing the dimer interface.

    “First, a buried Na+ ion occupying a unique position on the twofold axis invites us to view this cation as an intrinsic structural element stabilizing the dimer interface.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The antiparallel transmembrane topology of Fluc channels was established definitively by two-sided block of single channels by monobodies.

    “was established definitively by two-sided block of single channels by “monobodies,” engineered proteins selected as high-affinity binding partners from combinatorial libraries6.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [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) source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Bpe constructs carried two functionally neutral mutations to enhance expression, R29K/E94S or, for Hg labeling, R29K/E94C.

    “Bpe constructs carried two functionally neutral mutations to enhance expression, R29K/E94S or, for Hg labeling, R29K/E94C.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Ec2 constructs bore a single functionally neutral, expression-enhancing mutation, R25K, and an additional methionine A51M for selenomethionine incorporation.

    “Ec2 constructs bore a single functionally neutral, expression-enhancing mutation, R25K, and, for selenomethionine incorporation, an additional methionine was introduced (A51M) to enhance phasing power.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The S7 monobody was selected from a library designed to target convex protein surfaces.

    “This monobody was selected from a library designed to target convex protein surfaces14”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [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) source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Interpretationreasoning

    Because the pathways are extremely narrow, protein dynamics may be necessary to allow F- permeation, and the monobodies might force a conformation in which the two pores are less 'open' than in the fully conducting state.

    “Because these pathways are extremely narrow, protein dynamics may be necessary to allow F- permeation and of course the monobodies might force a conformation in which the two pores are less ‘open’ than in the fully conducting state.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Fluc proteins are ion channels with high specificity (>10^4) for F- over Cl- and a dual-topology dimeric architecture.

    “We recently established5,6 that Fluc proteins are ion channels with two unusual properties: an exceedingly high specificity (>104) for F- over Cl–, and a dual-topology dimeric architecture”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [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) source
    • cites [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) source

  • It may state two separate claims as one.

    Premisecitation

    Dual-topology dimeric construction is known in small multidrug transporters but has not been previously observed in ion channels.

    “Dual-topology dimeric construction is known in small multidrug transporters9–11 but has not been previously observed in ion channels.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [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) source
    • cites [10] Schuldiner, S. EmrE, a model for studying evolution and mechanism of ion-coupled transporters. Biochim. Biophys. Acta 1794, 748–762 (2009) source
    • cites [11] Morrison, E. A. et al. Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481, 45–50 (2011) source

  • It may state two separate claims as one.

    Premisecitation

    Recent experiments show that mutations of several C-terminal domain residues in pore-2 produce F- hypersensitivity in yeast, whereas mutations of N-terminal domain, pore-1 counterparts are relatively harmless.

    “Recent experiments buttress this idea by showing that mutations of several C-terminal domain residues in pore-2 produce F- hypersensitivity in yeast, whereas mutations of N-terminal domain, pore-1 counterparts are relatively harmless4.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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) source

  • It may state two separate claims as one.

    Premisecitation

    The double-pore assembly idea does not clash with any electrophysiological experiments, and double-pore assembly was explicitly cited previously as a possible though unlikely architecture consistent with the channel's functional behaviour.

    “Though unexpected, this idea does not clash with any electrophysiological experiments, and double-pore assembly was explicitly cited previously as a possible though unlikely architecture consistent with the channel’s functional behaviour5.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [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) source

  • It may state two separate claims as one.

    Study design and conductown data

    The Bpe-S7 structure was solved with phases initially determined by SAD of Hg-labelled Bpe and improved with Hg-labelled selenomethionine-substituted samples.

    “A structure was solved, with phases initially determined by single-wavelength anomalous diffraction (SAD) of Bpe labelled with Hg at a unique cysteine residue, and improved with Hg-labelled selenomethionine-substituted samples”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    This type of coordination is reminiscent of the phenylalanine rings of a proposed Cl--binding site in the bestrophin channel.

    “This type of coordination is reminiscent of the phenylalanine rings of a proposed Cl--binding site in the bestrophin channel21.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [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) source

  • It may state two separate claims as one.

    Premisecitation

    Ec2 is a homologue of only 33% identity, shows similar electrophysiological behavior to Bpe, and is blocked by S9 at nanomolar concentrations.

    “This homologue of only 33% identity shows similar electrophysiological behavior to Bpe and is blocked by S9 at nanomolar concentrations6.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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) source

  • It may state two separate claims as one.

    Interpretationown data

    The two F- ions of each pair lie in a vertical line tilted slightly off normal to the membrane plane, and so might mark a narrow permeation pathway.

    “The two F- ions of each pair lie in a vertical line tilted slightly off normal to the membrane plane, and so might mark a narrow permeation pathway.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    With a deficit of H-bond acceptors, the coordination shells observed here would be chemically inimical to ordered waters, which cannot be distinguished from F- based on x-ray scattering alone.

    “With a deficit of H-bond acceptors, the coordination shells observed here would be chemically inimical to ordered waters, which cannot be distinguished from F- based on x-ray scattering alone.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationcitation

    Two-pore assembly neatly accounts for evolutionary drift in eukaryotic Fluc channels, all of which consist of an inverted repeat of two homologous Fluc domains fused into a single polypeptide.

    “Two-pore assembly neatly accounts for evolutionary drift in eukaryotic Fluc channels, all of which consist of an inverted repeat of two homologous Fluc domains fused into a single polypeptide4,5.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [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) source
    • cites [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) source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Edge-on aromatic liganding of anions is rare but not unprecedented in proteins, and F- coordination by aromatic edges appears in many small-molecule structures.

    “Edge-on aromatic liganding of anions is rare but not unprecedented in proteins19, and F- coordination by aromatic edges appears in many small-molecule structures20.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [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) source
    • cites [20] Allen, F. H. The Cambridge Structural Database: a quarter of a million crystal structures and rising. Acta Crystallogr. B 58, 380–388 (2002) source

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    Coordination by only four oxygen ligands is uncommon for Na+ but is seen in ~15% of Na+-binding sites in the protein database.

    “Although coordination by only four oxygen ligands is uncommon for Na+, it is nevertheless seen in ~15% of Na+-binding sites in the protein database18.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Lev, B. R. B. & Noskov, S. Y. in Encyclopedia of Metalloproteins (eds Kretsinger, R. H. et al.) 2112–2118 (Springer, 2013) source

  • It may state two separate claims as one.

    Interpretationreasoning

    The pore, though narrow, is lined with H-bonding residues and so could provide a polar conduit for the ion's journey across the membrane span.

    “The pore, though narrow, is lined with H-bonding residues and so could provide a polar conduit for the ion’s journey across the membrane span.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    Flucs are double-barrelled F- channels, with the observed F- ions marking the ion-selective pathways.

    “This inference makes it difficult to resist the conclusion that Flucs are double-barrelled F- channels, with the observed F- ions marking the ion-selective pathways.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    To avoid biasing the model, NCS was not employed except at the final round of refinement to improve model geometry.

    “To avoid biasing the model, NCS was not employed except at the final round of refinement to improve model geometry.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    No prior phase information was used during refinement of the Bpe-S7 model.

    “No prior phase information was used during the refinement, however refinement was improved following anisotropic truncation of the structure factors.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Each value in Extended Data Table 2 represents mean ± s.e. of 3 determinations calculated from initial efflux rate.

    “Each value represents mean ± s.e. of 3 determinations calculated from initial efflux rate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductnone given

    Channels were recorded in the presence of 300 mM N-methyl-glucamine-fluoride, from which all small cations were rigorously excluded.

    “Channels were recorded in the presence of 300 mM N-methyl-glucamine-fluoride, from which all small cations were rigorously excluded.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductcitation

    Model validation for the Bpe-S7 structure was carried out using the Molprobity server.

    “Model validation was carried out using the Molprobity server44.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

57 of 57

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • FEX proteins are structurally related to Flucs but possess a more complex two-domain fold.

    “FEX proteins are related structurally to the Flucs but possess a more complex two-domain fold83,87.”

    From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [83]

  • Flucs assemble as unusual antiparallel dimers, with one protomer facing out and the other facing in.

    “The Flucs assemble as unusual antiparallel dimers, where one protomer is inserted into the membrane facing out, and the other is inserted into the membrane facing in81,83.”

    From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [83]

  • E88 is extremely well-conserved among fluoride channels and follows the same pattern of conservation/degradation in the different domains of the eukaryotic channels as other pore-lining residues.

    “This glutamate is extremely well-conserved among fluoride channels and follows the same pattern of conservation/degradation in the different domains of the eukaryotic channels as other pore-lining residues(73).”

    From Membrane exporters of fluoride ion (2021) · cites this paper as [73]

  • Mutation of either F82 or F85 to a non-polarizable hydrophobic residue like isoleucine greatly reduces or abolishes F− transport, depending on the homologue.

    “Mutation of either F82 or F85 to a non-polarizable hydrophobic residue like isoleucine greatly reduces or abolishes F− transport, depending on the homologue(73, 76).”

    From Membrane exporters of fluoride ion (2021) · cites this paper as [73]

  • In heterodimeric Fluc and FEX proteins, essential pore-lining residues are conserved in only one of the two domains.

    “In the heterodimeric Fluc and FEX proteins, essential pore-lining residues are conserved in only one of the two domains(3, 66, 73), providing a case study in how redundant features degrade upon gene duplication, fusion, and genetic drift.”

    From Membrane exporters of fluoride ion (2021) · cites this paper as [73]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • pH-activity statement cites the wrong extended-data figure

    Minor

    The statement points to Extended Data Figure 5 for F85I activity at different pH values, but Figure 5 describes Ec2-S9 electron density. The F85I pH comparison is described under Extended Data Figure 6.

  • Bpe-L2 data-collection resolution conflicts with the refinement resolution

    Minor

    The Bpe-L2 data-collection entry gives an overall resolution limit of 2.5 Å, although its parenthetical highest-resolution shell extends to 2.1 Å. The refinement entry for the same structure also gives a 2.1 Å limit.

2 of 2

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Metadata

Authors
Stockbridge, R. B., Kolmakova-Partensky, L., Shane, T., Koide, A., Koide, S., Miller, C., & Newstead, S.
Affiliations
  • Howard Hughes Medical Institute (US)
  • University of Chicago (US)
  • University of Oxford (GB)
Corresponding author
Newstead, Simon
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2015-09
Volume
525
Issue
7570
Pages
548-551
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/nature14981
PMC
PMC4876929
OpenAlex
W1893822674
Cited by
119
References
42
Citations by year
2026: 6 · 2025: 6 · 2024: 14 · 2023: 6 · 2022: 11 · 2021: 14 · 2020: 14 · 2019: 6 · 2018: 15 · 2017: 15 · 2016: 9 · 2015: 3
FWCI
4.96
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Wellcome Trust (102890/Z/13/Z, 102890)
  • National Institute of General Medical Sciences (K99GM111767, R01GM107023, U54GM087519)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
anion selectivity, anion coordination, fluoride toxicity, sodium cation coordination, crystal structure
MeSH terms
Anions, Bacterial Proteins, Cell Membrane, Fluorides, Ion Channels, Models, Biological, Models, Molecular, Phenylalanine, Protein Conformation, Crystallography, X-Ray

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-11-10, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-10 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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