Crystal structures of a double-barrelled fluoride ion channel

In the paper

“This monobody was selected from a library designed to target convex protein surfaces14”

What the sources say

• Partly supported

  The Results (Library Design; Crystal structures of monobody-target complexes confirm library designs) describe a side-and-loop library intended to recognize convex surfaces and report that SH13, a monobody from that library, binds a convex Abl SH2 surface. Our copy does not identify S7 as a monobody selected from that library, so it supports the library-design point but not the claim about S7 specifically. (LLM, conf=0.84)

  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) sourceExperimental — accuracy not yet measured

In the paper

“however diffraction to 3.6 Å Bragg spacing was obtained with monobody Mb(Bpe-S7)6, hereafter denoted S7.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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).”

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [22] Walden, M. et al. Uncoupling and turnover in a Cl−/H+ exchange transporter. J. Gen. Physiol. 129, 317–329 (2007) source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  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

In the paper

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

In the paper

“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.”

What the sources say

• ?Not matched

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

  cites SHELX

In the paper

“For F85I and F82I, efflux of F- is 2 and 3 orders of magnitude slower, respectively, than for wild type (~3x105 s-1)15”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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”

In the paper

“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.”

In the paper

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

In the paper

“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.”

What the sources say

• ?Not matched

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

  cites SHELX36
• ?Not matched

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

  cites SHARP37
• ?Not matched

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

  cites SOLOMON38

In the paper

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

In the paper

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

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [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

In the paper

“Phases were calculated using Se-SAD with 8 Se sites, and processed as above.”

In the paper

“For the Bpe-L2 crystals, data were similarly processed and scaled in spacegroup P1.”

In the paper

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

In the paper

“For the Ec2-S9 crystals the data were processed as above with space group P41.”

In the paper

“The electron density maps clearly showed major differences in the selected, variable regions of the monobody.”

In the paper

“No prior phase information was used during the refinement, however refinement was improved following anisotropic truncation of the structure factors.”

In the paper

“The experimental electron density maps were of exceptional quality following phase extension to the highest resolution shell of 2.58 Å.”

In the paper

“microorganisms export this anion through F--specific ion channels of the Fluc family1–4.”

What the sources say

• Not checked yetcites [1] Baker, J. L. et al. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235 (2012) source
• Not checked yetcites [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
• Not checked yetcites [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
• Not checked yetcites [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

In the paper

“including extreme selectivity for F- over Cl- and dual-topology dimeric assembly5–6.”

What the sources say

• Not checked yetcites [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
• Not checked yetcites [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

In the paper

“The fluoride anion, ubiquitous in the aqueous biosphere throughout evolutionary time, is a xenobiotic inhibitor of essential phosphoryl-transfer enzymes7.”

What the sources say

• ?Not matched

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

In the paper

“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.”

In the paper

“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”

In the paper

“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.”

In the paper

“was established definitively by two-sided block of single channels by “monobodies,” engineered proteins selected as high-affinity binding partners from combinatorial libraries6.”

What the sources say

• Not checked yetcites [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

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

“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”

What the sources say

• Not checked yetcites [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
• Not checked yetcites [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

In the paper

“Dual-topology dimeric construction is known in small multidrug transporters9–11 but has not been previously observed in ion channels.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  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
• ?Not checked — no full text of the sourcecites [10] Schuldiner, S. EmrE, a model for studying evolution and mechanism of ion-coupled transporters. Biochim. Biophys. Acta 1794, 748–762 (2009) source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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”

In the paper

“This type of coordination is reminiscent of the phenylalanine rings of a proposed Cl--binding site in the bestrophin channel21.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  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

In the paper

“This homologue of only 33% identity shows similar electrophysiological behavior to Bpe and is blocked by S9 at nanomolar concentrations6.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

In the paper

“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.”

What the sources say

• Not checked yetcites [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
• Not checked yetcites [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

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [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
• ?Not checked — no full text of the sourcecites [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

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [18] Lev, B. R. B. & Noskov, S. Y. in Encyclopedia of Metalloproteins (eds Kretsinger, R. H. et al.) 2112–2118 (Springer, 2013) source

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

“No prior phase information was used during the refinement, however refinement was improved following anisotropic truncation of the structure factors.”

In the paper

“Each value represents mean ± s.e. of 3 determinations calculated from initial efflux rate.”

In the paper

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

In the paper

“Model validation was carried out using the Molprobity server44.”

What the sources say

• ?Not matched

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

  cites [44]