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Cited by 65Toxic Organic Pollutants ImpactProtein Structure and DynamicsComputational Drug Discovery Methods

Comparative Analysis of Homology Models of the Ah Receptor Ligand Binding Domain: Verification of Structure–Function Predictions by Site-Directed Mutagenesis of a Nonfunctional Receptor

Open access
Fraccalvieri, Domenico, Soshilov, Anatoly A., Karchner, Sibel I., Franks, Diana G., Pandini, Alessandro, Bonati, Laura, Hahn, Mark E., Denison, Michael S.·2013·Biochemistry
DOI 10.1021/bi301457fPMC PMC3568667
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Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness.

AI snapshot

Written from the paper’s full text.

TL;DR

This study built and compared homology models of the aryl hydrocarbon receptor ligand-binding domain for sixteen AHRs from twelve species, then used site-directed mutagenesis to test predictions about why zebrafish AHR1a cannot bind TCDD. Two substitutions in zfAHR1a restored TCDD binding and TCDD-dependent DNA binding, but not TCDD-dependent transcriptional activation.

Key findings

  • Homology models of sixteen AHR ligand-binding domains from twelve mammalian, avian, and fish species revealed a conserved overall fold, with modeled internal cavities ranging from about 300 to 600 ų.
  • The zfAHR1a cavity was calculated at roughly 300 ų and appeared truncated, whereas the high-affinity zfAHR2 cavity was about 600 ų and spanned the domain fold; three zfAHR1a residues (Tyr296, Thr386, His388) were predicted to sterically restrict TCDD access.
  • Mutating zfAHR1a at Y296H or T386A partially restored specific [³H]TCDD binding, and the Y296H+T386A double mutant fully restored binding, with 2- to 3-fold more binding than zfAHR1b or zfAHR2; H388Q did not increase binding and reduced restoration when combined with T386A.
  • The Y296H+T386A double mutant and the Y296H+T386A+H388Q triple mutant showed TCDD-dependent DNA binding, with the triple mutant lower than the double mutant, but neither wild-type nor mutant zfAHR1a produced TCDD-induced luciferase activity in COS-7 cells.

Methods

  • Homology models were generated for sixteen AHR PAS-B ligand-binding domains using HIF-2α and ARNT NMR structures as templates, MODELLER for model building, PROCHECK and ProSA for validation, DSSPcont for secondary structure, and CASTp for cavity identification and volume calculation.
  • Zebrafish AHR1a mutants were created by site-directed mutagenesis at positions 296, 386, and 388, expressed in vitro, and tested for [³H]TCDD binding by sucrose gradient centrifugation and for TCDD-dependent DNA binding by gel retardation with zfARNT1c.
  • Transcriptional activation was assayed in COS-7 cells transiently transfected with wild-type or mutant zfAHRs, ARNT2b, the pGudLuc6.1 reporter, and a Renilla control, then treated with DMSO or 10 nM TCDD.

Limitations

  • The paper states that homology modeling gives somewhat inaccurate internal side-chain conformations, so cavity volume alone cannot reliably predict ligand accessibility; it also notes that the mechanism behind increased binding by the Y296H+T386A mutant is unclear and that whether these mutations increase affinity or the number of functional ligand-binding AHRs remains undetermined.
  • The paper states that zfAHR1a’s transactivation domain appears nonfunctional in these assays, and that zfAHR1a may still respond to structurally distinct ligands such as leflunomide, leaving its true ligand selectivity unresolved.
  • Evident from the text: experimental validation focused on TCDD and zebrafish AHR1a, the models are theoretical rather than experimentally determined structures, and the study does not establish whether the same predictions hold for other AHRs or ligands.

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

Integrity signals

Registry

Crossref · 10.1021/bi301457f

Cites

63 of 63 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

61 of 114 cited sources checked · 33 supported · 2 flagged · accuracy not yet measured

  • Premisereasoning

    Experimental confirmation of predicted binding modes for TCDD and other ligands in the AHR LBD is generally lacking.

    “While the binding modes of TCDD and other ligands within the AHR LBD of a given species (typically that of mouse or human AHR) have been predicted by these approaches, experimental confirmation of these proposed interactions are generally lacking.”
    Quote in the paperSubstantial claim5 of 8 sources checked
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [18] Pandini A., 2009, Biochemistry source

      The Abstract and Discussion report experimental validation of predicted TCDD-binding residues through site-directed mutagenesis and functional analysis. The paper therefore provides experimental support for proposed TCDD interactions rather than saying such confirmation is lacking. It does not establish how often binding modes for other ligands have been confirmed. (LLM, conf=0.83)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [19] Motto I., 2011, J. Chem. Inf. Model. source

      The Introduction says the predicted TCDD binding mode was confirmed by functional analysis of mouse AHR mutants. The Results and Discussion section reports new mutagenesis data that “further confirm” that binding mode. Although the Conclusions say no experimentally determined AHR LBD structures were available, that is not the same as saying experimental confirmation of proposed interactions was generally lacking. (LLM, conf=0.84)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [20] Bisson W. H., 2009, J. Med. Chem. source

      The paper’s modeling discussion (page unavailable in our copy) predicts TCDD docking orientations in mouse, human, and zebrafish AhR-LBD models. It also reports cell-based tests of predicted ligands, but these test activity rather than directly confirming their proposed binding modes. The paper does not state the broader claim that experimental confirmation of such interactions is generally lacking. (LLM, conf=0.80)

      Experimental — accuracy not yet measured
    • cites [22] O’Donnell E. F., 2010, PLoS One source

      The Molecular Docking Studies and Molecular Docking of Leflunomide and A771726 sections (pages unavailable) describe predicted binding interactions in mouse and human AhR ligand-binding-domain models. They do not address whether experimental confirmation of binding modes for TCDD and other ligands is generally lacking; the reported cellular assays test receptor activity, not the proposed structural interactions. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [23] Wu B., 2009, Toxicol. Lett. source
    • cites [24] Jogalekar A. S., 2010, Bioorg. Med. Chem. Lett. source
    • cites [25] Murray I. A., 2011, Mol. Pharmacol. source

      The Results section, “DiMNF Adopts a Unique Orientation within the Ligand Binding Pocket of AHR” (page unavailable), presents predicted binding interactions in mouse and human AHR. The Discussion says mutational analysis may verify a predicted DiMNF interaction and that a crystal structure may clarify ligand-induced differences. These passages support a lack of confirmation for the paper’s proposed DiMNF interaction, but do not establish that experimental confirmation of binding modes for TCDD and other ligands is generally lacking. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    Homology models of the AHR ligand-binding domain exist for only a small number of species.

    “homology models of the AHR ligand-binding domain (LBD) are available for only a few species.”
    • Also stated in: § INTRODUCTION · plain
      “modeling studies have been focused on a limited number of species (12,17-20).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [17] Pandini A., 2007, Biochemistry source

      The Homology Modeling section describes a model of the mouse AhR ligand-binding domain, and the paper also discusses earlier mouse models. It does not establish how many species have such models or state that models are available for only a few species; no page number is available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [18] Pandini A., 2009, Biochemistry source
    • cites [19] Motto I., 2011, J. Chem. Inf. Model. source

      The Introduction (page unavailable) describes AhR LBD homology models for mouse, human, and rat and refers to models of several mammalian AhRs. It does not establish that models are available for only a few species overall. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [20] Bisson W. H., 2009, J. Med. Chem. source

      The model-construction passage (section labeled Abstract; page not provided) describes AHR-LBD homology models for mouse, human, rat, and two zebrafish isoforms. That supports the existence of models for a small set of species, but the paper does not establish that models are available for only a few species overall. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Premisecitation

    The authors and others have used crystal and NMR structures of homologous protein domains to build homology models of the AHR LBD.

    “While the lack of any three-dimensional structure information on the AHR ligand binding domain (LBD) has prevented detailed molecular analysis of the mechanisms of AHR ligand binding, we and others have used the availability of crystal and nuclear magnetic resonance (NMR) structures of homologous protein domains to develop homology models of the AHR LBD for such analysis (12,17-20).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [17] Pandini A., 2007, Biochemistry source
    • cites [18] Pandini A., 2009, Biochemistry source

      The Materials and Methods section (Homology Modeling; page unavailable) says the authors used NMR structures of homologous HIF-2α and ARNT PAS B domains as templates for AhR LBD homology models. The paper also mentions earlier mouse AhR LBD models, but the passages found do not establish the claim's crystal-structure component. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [19] Motto I., 2011, J. Chem. Inf. Model. source
    • cites [20] Bisson W. H., 2009, J. Med. Chem. source
  • Premisecitation

    Zebrafish have three AHRs: zfAHR1a, zfAHR1b, and zfAHR2.

    “Zebrafish (Danio rerio) possess three AHRs, one of which (AHR1a) has been shown previously to lack the ability to bind TCDD and DNA and to activate transcription in the presence of TCDD [28,29].”
    • Also stated in: § Selection of AHR sequences · plain
      “the zebrafish contains three distinct AHRs (zfAHR1a, zfAHR1b and zfAHR2 (28,29,49)).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [28] Andreasen E. A., 2002, Mol. Pharmacol. source
    • cites [29] Karchner S. I., 2005, Biochem. J. source
    • cites [49] Tanguay R. L., 1999, Biochim. Biophys. Acta source
  • Premisecitation

    The absence of three-dimensional structure information for the AHR LBD has prevented detailed molecular analysis of AHR ligand binding mechanisms.

    “While the lack of any three-dimensional structure information on the AHR ligand binding domain (LBD) has prevented detailed molecular analysis of the mechanisms of AHR ligand binding, we and others have used the availability of crystal and nuclear magnetic resonance (NMR) structures of homologous protein domains to develop homology models of the AHR LBD for such analysis (12,17-20).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [17] Pandini A., 2007, Biochemistry source
    • cites [18] Pandini A., 2009, Biochemistry source
    • cites [19] Motto I., 2011, J. Chem. Inf. Model. source
    • cites [20] Bisson W. H., 2009, J. Med. Chem. source

      The Experimental Section (Homology Modeling; page unavailable) says the authors used an NMR structure of a homologous domain as a template for AhR-LBD models, supporting the need to model its structure. The paper does not say that the lack of an AhR-LBD structure prevented detailed molecular analysis; its Abstract instead reports structural analyses made using those models. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • May combine two claims. 39
  • Classified as probably not a substantial claim. 27

  • May combine two claims.

    Premisecitation

    Substantial differences among species in AHR ligand binding specificity, selectivity, and response have been observed, but the structural determinants behind them remain unknown.

    “While significant interspecies differences in AHR ligand binding specificity, selectivity and response have been observed, the structural determinants responsible have not been determined and homology models of the AHR ligand-binding domain (LBD) are available for only a few species.”
    • Also stated in: § INTRODUCTION · plain
      “dramatic intra- and inter-species differences that have been observed in AhR ligand binding, ligand selectivity and response (3,10,14,26,27).”
    Quote in the paperSubstantial claim2 of 5 sources checked
    • cites [3] Denison M. S., 2011, Toxicol. Sci. source

      The sections on AhR ligand binding and species-specific differences report interspecies differences in ligand binding and response, and describe LBD models for several species (pages unavailable). But the species-specific differences section also says mutagenesis has identified amino acids contributing to some of those differences, so the claim that the structural determinants remain wholly unknown overstates the paper. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [10] Poland A., 1982, Annu. Rev. Pharmacol. Toxicol. source
    • cites [14] DeGroot D., 2011 source
    • cites [26] Flaveny C. A., 2009, Mol. Pharmacol. source

      The ligand-binding results report different affinities and responses for human and mouse AHR. The Discussion says the residues critical to high-affinity human AHR binding of indirubin require further investigation, but the Abstract attributes the known human–mouse difference in TCDD affinity to Val381; the claim overstates how little is known about the structural determinants. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [27] Aarts J. M., 1995, Eur. J. Pharmacol. source

  • Classified as probably not a substantial claim.

    Premisecitation

    The aryl hydrocarbon receptor is a ligand-activated transcription factor that mediates the biological and toxic effects of many structurally diverse chemicals, including TCDD.

    “The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [1] Beischlag T. V., 2008, Crit. Rev. Eukaryotic Gene Expression source
    • cites [2] Furness S. G., 2009, Pharmacol. Ther. source
    • cites [3] Denison M. S., 2011, Toxicol. Sci. source

  • May combine two claims.

    Premisecitation

    The tern AHR has about 4- to 6-fold lower TCDD affinity than high-affinity mouse and chicken AHRs, but is classified as medium/low because its TCDD binding is sensitive to detergent washes.

    “The tern AHR also demonstrated an approximately 4- to 6-fold lower TCDD binding affinity than that of high affinity mAHR and chicken AHR, but since [3H]TCDD binding to this AHR was particularly sensitive to washes with detergent-containing buffer (54), it was classified as a ‘medium/low’ affinity AHR (Table 1).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [54] Karchner S. I., 2006, Proc. Natl. Acad. Sci. U.S.A. source

      The Results (page not provided) report lower TCDD-binding affinity for tern than chicken AHR and say the tern AHR–TCDD complex is more easily disrupted by HAP-assay washes. They report a 7-fold difference from chicken, not 4- to 6-fold; they do not establish that ratio relative to mouse AHR or classify tern AHR as “medium/low” because of sensitivity to detergent-containing washes. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The biological and toxic effects of ligand-activated AHRs depend on the physicochemical properties and metabolic persistence of the ligand.

    “the spectrum of biological and toxic effects produced by the ligand-activated AHRs is dependent upon the physicochemical characteristics and metabolic persistence of the ligand (3,7,10).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [3] Denison M. S., 2011, Toxicol. Sci. source
    • cites [7] Safe S., 1990, Crit. Rev. Toxicol. source
    • cites [10] Poland A., 1982, Annu. Rev. Pharmacol. Toxicol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Ligand binding to the cytosolic AHR stimulates its translocation to the nucleus, and dimerization with ARNT converts the ligand:AHR:ARNT complex into a high-affinity DNA-binding form.

    “Mechanistically, binding of ligand to the AHR in the cytosol of a cell stimulates translocation of the AHR protein complex into the nucleus and its subsequent dimerization with the ARNT (AHR nuclear translocator) protein transforms the ligand:AHR:ARNT complex into its high-affinity DNA binding form (3-5).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [3] Denison M. S., 2011, Toxicol. Sci. source
    • cites [4] Soshilov A., 2008, J. Biol. Chem. source
    • cites [5] Hankinson O., 1995, Annu. Rev. Pharmacol. Toxicol. source

  • May combine two claims.

    Premisecitation

    Homology models of the AHR LBD have revealed key structural features and, when combined with site-directed mutagenesis and functional assays, have helped identify amino acids important for ligand binding and ligand-dependent activation.

    “These theoretical models not only have revealed key structural aspects of the domain, but when coupled with site directed mutagenesis and functional analysis, have proven to be helpful in identifying amino acids important in ligand binding and ligand-dependent AHR activation (12,17-19,21).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [17] Pandini A., 2007, Biochemistry source
    • cites [18] Pandini A., 2009, Biochemistry source
    • cites [19] Motto I., 2011, J. Chem. Inf. Model. source
    • cites [21] Soshilov A., 2011, J. Biol. Chem. source

  • May combine two claims.

    Premisecitation

    zfAHR1a does not detectably bind TCDD, bind DNA, or activate transcription in response to TCDD.

    “one of which (AHR1a) has been shown previously to lack the ability to bind TCDD and DNA and to activate transcription in the presence of TCDD [28,29].”
    • Also stated in: § Selection of AHR sequences · boosted
      “zfAHR1a did not demonstrate any detectable [3H]TCDD binding nor transcriptional response to TCDD (28,29).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [28] Andreasen E. A., 2002, Mol. Pharmacol. source
    • cites [29] Karchner S. I., 2005, Biochem. J. source

      The Functional characterization section reports that AHR1A failed to bind TCDD and did not activate a reporter above the no-AHR control, including after TCDD exposure. Our copy does not establish the separate claim that AHR1A cannot bind DNA. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    zfAHR1b has lower TCDD affinity than zfAHR2 and is classified as medium affinity based on an approximately 8-fold lower potency for TCDD-dependent transcription activation in COS-7 cells.

    “However, based on the approximately 8-fold lower potency of TCDD-dependent activation of transcription in COS-7 cells transiently transfected with zfAHR1b, compared to those containing zfAHR2, zfAHR1b is designated as having a ‘medium’ TCDD-binding affinity (29).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Karchner S. I., 2005, Biochem. J. source

      The Functional characterization section reports that TCDD-dependent transcriptional activation in COS-7 cells had apparent EC50 values of 5.9 nM for AHR1B and 0.7 nM for AHR2—approximately an eightfold difference. The Functional implications section also says AHR1B has somewhat lower TCDD-binding affinity than AHR2. However, the paper does not designate AHR1B’s affinity as “medium”; it describes its TCDD binding as high-affinity. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Reported TCDD binding affinity estimates for the C57BL/6 mouse AHR range from 6 pM to 2.4 nM, which is considered high affinity.

    “Accordingly, TCDD binding affinity estimates for the C57BL/6 mAHR, which is used as the basis for comparisons, are reported to range between 6 pM – 2.4 nM; this is considered to be high affinity (53-58).”
    Quote in the paperNot a substantial claim1 of 6 sources checked
    • cites [53] Ema M., 1994, J. Biol. Chem. source
    • cites [54] Karchner S. I., 2006, Proc. Natl. Acad. Sci. U.S.A. source

      The Results report high-affinity TCDD binding by mouse AHR, with a K_D of 2.39 ± 0.80 nM (Fig. 2A), supporting the approximately 2.4 nM endpoint. Our copy does not report a 6 pM estimate or identify the mouse AHR as specifically from C57BL/6 mice. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [55] Jensen B. A., 2001, Toxicol. Sci. source
    • cites [56] Denison M. S., 1986, Chemosphere source
    • cites [57] Kim E. Y., 2002, Aquat. Toxicol. source
    • cites [58] Poland A., 1994, Mol. Pharmacol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Binding of the ligand-activated AHR complex to the dioxin responsive element stimulates transcription of adjacent genes and mediates the toxic and biological effects of AHR ligands.

    “Binding of the ligand-activated AHR complex to its specific DNA recognition site, the dioxin responsive element (DRE), stimulates transcription of adjacent genes and thus mediates the toxic and biological effects of AHR ligands (1,3).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [1] Beischlag T. V., 2008, Crit. Rev. Eukaryotic Gene Expression source
    • cites [3] Denison M. S., 2011, Toxicol. Sci. source

      The “Classical Mechanism of AhR Action” section (page not provided) says that binding of the ligand:AhR:ARNT complex to a DRE increases gene transcription. It says this mechanism is suggested to account for many toxic and biological effects, but does not establish that DRE-driven transcription mediates all such effects; the paper also discusses nonclassical mechanisms. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The ligand binding promiscuity of a given AHR may result from differential binding of structurally diverse ligands within its ligand-binding cavity.

    “More recent studies have reported that the ligand binding promiscuity of a given AHR may result from differential binding of structurally diverse ligands within the AHR ligand-binding cavity (11-14), similar to the mechanisms responsible for pregnane X receptor ligand binding promiscuity (15,16).”
    Quote in the paperSubstantial claim1 of 4 sources checked
    • cites [11] Whelan F., 2010, Mol. Pharmacol. source
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [13] Zhao B., 2010, Toxicol. Sci. source
    • cites [14] DeGroot D., 2011 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The AHR is a ligand-dependent bHLH/PAS transcription factor that regulates gene expression and other cellular signaling events.

    “The aryl hydrocarbon receptor (AHR) is a ligand-dependent basic helix-loop-helix (bHLH)/Per-ARNT-Sim (PAS) domain-containing transcription factor that regulates gene expression and other cellular signaling events (1-3).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] Beischlag T. V., 2008, Crit. Rev. Eukaryotic Gene Expression source
    • cites [2] Furness S. G., 2009, Pharmacol. Ther. source
    • cites [3] Denison M. S., 2011, Toxicol. Sci. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    This volume range is within the 100-800 Å3 range commonly seen in protein binding pockets or cavities.

    “which is within the range (100 - 800 Å3) commonly observed in protein binding pockets or cavities (62).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [62] Liang J., 1998, Protein Sci. source

      In “Pockets and cavities with known ligands” (page unavailable), the paper says most ligand-binding sites in its set of monomeric enzymes are on the order of 100–1,000 ų. That supports the general scale, but not the more specific claim that 100–800 ų is the commonly observed range. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    Both substitutions involve residues inside the modeled cavities and belong to the mammalian TCDD-binding fingerprint.

    “Both these substitutions involve residues that are internal to the modeled cavities and that belong to the “TCDD-binding fingerprint” for mammalian AHRs (18).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Pandini A., 2009, Biochemistry source
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • Classified as probably not a substantial claim.

    Premisecitation

    A prior homology modeling study proposed that zfAHR1a cannot bind TCDD because its internal cavity residues differ from zfAHR2, reducing cavity volume and changing pocket polarity.

    “A previous homology modeling study suggested that the inability of zfAHR1a to bind TCDD was due to the presence of different internal cavity residues from those in zfAHR2 that decreased the volume and altered the polarity of the binding pocket itself (20).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Bisson W. H., 2009, J. Med. Chem. source

  • May combine two claims.

    Interpretationcitation

    In the model, a Val at position 375 creates steric hindrance at one end of the cavity, reducing cavity volume and potentially altering interactions with other fingerprint residues.

    “When this amino acid is mutated to Val, it introduces steric hindrance at this end of the modeled cavity (Figure 2C), effectively decreasing the cavity volume and potentially affecting ligand interactions with other fingerprint residues (17,18).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [17] Pandini A., 2007, Biochemistry source

      Experimental Testing and Model Validation says that replacing Ala375 with Val increases steric hindrance within the modeled cavity and reduces TCDD binding; the Conclusions say Val reduces ligand accessibility. However, the paper places residue 375 at the center of the cavity, not at one end, and does not specifically attribute altered interactions with other fingerprint residues to this mutation. Reduced available cavity space is consistent with its steric explanation, but a decrease in cavity volume is not reported directly. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [18] Pandini A., 2009, Biochemistry source

      The Discussion (page not available) describes increased steric hindrance at position 375 and reduced internal cavity space in modeled mutants; the Results on molecular docking also report weaker predicted TCDD binding for A375V. The paper describes position 375 as central, not at one end of the cavity, and these passages do not establish that Val alters ligand interactions with other fingerprint residues. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Premisecitation

    zfAHR2 and zfAHR1b bind TCDD with high and medium affinity, respectively, whereas zfAHR1a does not bind TCDD.

    “while zfAHR2 and zfAHR1b can bind TCDD with high and medium affinity, respectively, zfAHR1a fails to bind TCDD (28,29).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [28] Andreasen E. A., 2002, Mol. Pharmacol. source
    • cites [29] Karchner S. I., 2005, Biochem. J. source

      The Functional characterization section reports high-affinity TCDD binding by AHR2 and no binding by AHR1A. It finds that AHR1B binds TCDD with somewhat lower affinity than AHR2, but explicitly describes AHR1B’s binding as high-affinity, not medium-affinity. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Three zebrafish AHRs have been described: zfAHR1a, zfAHR1b, and zfAHR2.

    “Three zebrafish AHRs have been described (zAHR1a, zfAHR1b and zfAHR2)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [28] Andreasen E. A., 2002, Mol. Pharmacol. source
    • cites [29] Karchner S. I., 2005, Biochem. J. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hydrophobic stabilization by the long Ile319 side chain (position 324/325 in ch/tAHR) was previously proposed as necessary for optimal TCDD binding by mAHR.

    “Accordingly, hydrophobic stabilization by the long Ile319 side chain (corresponding to position 324/325 in the ch/tAHR) has been previously suggested to be necessary for optimal TCDD binding by mAHR (18).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Pandini A., 2009, Biochemistry source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Homology models have been used in molecular docking to study interactions of known agonists within the AHR LBD and in virtual screening of putative ligands.

    “More recently, these models have been used in molecular docking studies in an attempt to elucidate the detailed interactions of some known ligands (agonists) within the AHR LBD as well as in the virtual screening of collections of putative ligands (12,18-20,22-25).”
    Quote in the paperNot a substantial claim5 of 8 sources checked
    • cites [12] Xing Y., 2012, Toxicol. Sci. source
    • cites [18] Pandini A., 2009, Biochemistry source

      The Materials and Methods and Results sections on Molecular Docking describe using a homology model of the mouse AHR ligand-binding domain to dock TCDD and examine its binding interactions. Our copy does not report virtual screening of collections of putative ligands, so it supports the docking portion but not that portion of the claim. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [19] Motto I., 2011, J. Chem. Inf. Model. source
    • cites [20] Bisson W. H., 2009, J. Med. Chem. source
    • cites [22] O’Donnell E. F., 2010, PLoS One source

      The Molecular Docking Studies and Molecular Docking of Leflunomide and A771726 sections report docking the AhR agonist leflunomide into mouse and human AhR-LBD homology models to examine its interactions. Our copy does not report using those models to virtually screen collections of putative ligands; it describes that approach in the title of a separately cited study (reference 33). (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [23] Wu B., 2009, Toxicol. Lett. source
    • cites [24] Jogalekar A. S., 2010, Bioorg. Med. Chem. Lett. source
    • cites [25] Murray I. A., 2011, Mol. Pharmacol. source

      The Molecular Docking section and the section on DiMNF’s orientation in the AHR ligand pocket describe modeled docking of ligands and predicted interactions with specific residues. Our copy does not describe virtual screening of ligand collections or explicitly identify the model as a homology model; those parts of the citing sentence are not supported by this source. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The affinity difference between chAHR (high) and tAHR (medium/low) was previously attributed to two substitutions, I324/V325 and S380/A381.

    “Previously, the difference in TCDD binding affinity between the chAHR (high affinity for TCDD) and tAHR (medium/low affinity for TCDD) was attributed to two amino acid substitutions, I324/V325 and S380/A381 (54).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [54] Karchner S. I., 2006, Proc. Natl. Acad. Sci. U.S.A. source

  • Classified as probably not a substantial claim.

    Premisecitation

    A consensus cavity for high-affinity mammalian AHRs was previously defined as the cavity bounded by internal residues conserved across all modeled LBDs.

    “For the mammalian AHRs with relatively high affinity for TCDD (first seven AHRs in Table 1) a “consensus cavity” was previously defined as the cavity delimited by the internal residues (obtained by CASTp) that are conserved in all the modeled LBDs (18).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Pandini A., 2009, Biochemistry source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    One hsp90 binding site lies within the AHR LBD.

    “Given our recent results indicating that one of the binding sites of the AHR chaperone protein hsp90 is contained within the AHR LBD (21)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [21] Soshilov A., 2011, J. Biol. Chem. source

  • May combine two claims.

    Premisecitation

    TCDD stimulates gene expression through zfAHR2 and zfAHR1b but not through zfAHR1a.

    “As expected, TCDD can stimulate gene expression by zfAHR2 and zfAHR1b, but not by zfAHR1a (28,29).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [28] Andreasen E. A., 2002, Mol. Pharmacol. source
    • cites [29] Karchner S. I., 2005, Biochem. J. source

  • May combine two claims.

    Premisecitation

    Changing Ala375 to valine, or having valine naturally at this position in DBA mouse or human AHR (V381), lowers TCDD affinity.

    “since its mutation to valine (17,63) or natural occurrence as valine in the DBA mouse AHR or in the human AHR (V381) (53,58) results in a decreased affinity for TCDD.”
    Quote in the paperSubstantial claim1 of 4 sources checked
    • cites [17] Pandini A., 2007, Biochemistry source
    • cites [63] Murray I. A., 2005, Arch. Biochem. Biophys. source
    • cites [53] Ema M., 1994, J. Biol. Chem. source
    • cites [58] Poland A., 1994, Mol. Pharmacol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    For zfAHR DNA binding assays, zfARNT1c was used because preliminary analyses showed it produced more TCDD-dependent DNA binding than zfARNT2b.

    “For zfAHRs, DNA binding analysis was carried out using zfARNT1c because preliminary analyses revealed that it resulted in a greater amount of TCDD-dependent DNA binding than that obtained using zfARNT2b (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    For every AHR target sequence, the top sequence-similarity hits were the PASB domains of HIF-2α and ARNT.

    “For all targets, the top hits were the PASB domains of hypoxia-inducible factor 2α (HIF-2α) and ARNT.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the zfAHR2 and zfAHR1b models, most internal cavity residues are conserved and occupy corresponding positions, but a small group is differently positioned.

    “Comparison of the internal residues in the zfAHR2 and zfAHR1b modeled cavities (Figures 3A, B, C) indicates that, while most of these residues occupy corresponding positions and are conserved in the two AHRs, a small group lies in different positions.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Findingown data

    CASTp calculated the zfAHR1a cavity as about 300 Å3 with a truncated shape, compared with about 600 Å3 for zfAHR2, which spans the entire domain fold.

    “This was also confirmed by the smaller calculated volume (about 300 Å3) and truncated shape of this cavity determined by CASTp in comparison to the internal cavity in zfAHR2 (about 600 Å3) which spans the entire domain fold (Figure 3B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Findingown data

    Constitutive and TCDD-inducible DNA binding by zfAHR1b was much higher than by zfAHR2 or the double-mutant zfAHR1a, but the TCDD-dependent enhancement of DNA binding was quantitatively similar for zfAHR2 and zfAHR1b.

    “Interestingly, while the levels of both constitutive and TCDD-inducible DNA binding by zfAHR1b were dramatically higher than those of zfAHR2 or zfAHR1a containing the double mutation, quantitatively, the amounts of the TCDD-dependent enhancement of DNA binding by zfAHR2 and zfAHR1b were similar (Figure 5B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Findingown data

    The high-affinity fish AHR cavity has more internal residues and a more elongated shape at both ends than the mammalian/avian cavities.

    “In contrast, the cavity of the high affinity fish AHR is characterized by a higher number of internal residues (see Figure 3A), associated with a more elongated shape at both sides (delimited by the Fα helix on one side and the C-terminal β-strands on the other).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Findingown data

    A small but significant increase in TCDD-dependent DNA binding occurred with the Y296H mutant, but not with T386A or H388Q.

    “A small but significant increase in TCDD-dependent DNA binding was observed with zfAHR1a containing the Y296H mutation, but not that with T386A or H388Q.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Findingown data

    TCDD-dependent transformation and DNA binding occurred with zfAHR1a carrying Y296H/T386A and with the Y296H/T386A/H338Q triple mutant, though the triple mutant bound DNA less than the double mutant.

    “Similar to the ligand binding results described above, TCDD-dependent transformation and DNA binding was observed with zfAHR1a containing the Y296H/T386A double mutation as well as that containing the Y296H/T386A/H338Q triple mutation, with DNA binding by the triple mutant zfAHR1a lower than that of the double mutant.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Findingown data

    The Y296H and T386A single mutations each partially restored specific [3H]TCDD binding to zfAHR1a.

    “The results of the ligand binding analyses reveal that two of the single mutations, Y296H and T386A, resulted in partial restoration of specific [3H]TCDD binding to the zfAHR1a (Figure 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Findingown data

    The high-affinity mammalian consensus cavity and the chAHR cavity are very similar in shape and size and share the same internal conserved residues.

    “The consensus cavity for the high affinity mammalian and the chAHR cavity are very similar in shape and size and, as described below, they also share the same internal conserved residues (see Figure 2A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Findingown data

    The H388Q mutation did not increase [3H]TCDD binding; with T386A it reduced restoration, but it did not affect AHRs carrying Y296H.

    “No increase in [3H]TCDD specific binding was observed with the H388Q mutation and inclusion of this mutation had a negative effect on ligand binding restoration when combined with a T386A mutation, but no effect on AHRs containing the Y296H mutation (Figure 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Findingown data

    The Y296H+T386A double mutation fully restored specific [3H]TCDD binding to zfAHR1a, with binding 2- to 3-fold higher than for zfAHR1b or zfAHR2.

    “Interestingly, [3H]TCDD specific binding to zfAHR1a containing the Y296H+T386A double mutation was not only completely restored, but the amount of binding was 2- to 3-times greater than that observed for the fully functional zfAHR1b or zfAHR2.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Findingown data

    TCDD induced luciferase activity with wild-type zfAHR1b or zfAHR2, but not with wild-type or mutant zfAHR1a, regardless of its TCDD binding or DNA binding.

    “While TCDD induced luciferase activity in cells transfected with wild-type zfAHR1b or zfAHR2, no TCDD-induction was observed in cells cotransfected with wild-type or mutant zfAHR1a, irrespective of its ability to bind TCDD or exhibit TCDD-dependent DNA binding (Figure 6).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The COS-7 luciferase reporter assay reflects the transactivation function of zfAHR1a in vivo.

  • May combine two claims.

    Study design and conductown data

    The models had ProSA z-scores between -3.29 and -5.25, which fall within the range seen for native protein structures of similar size.

    “Moreover the ProSA z-scores were between -3.29 and -5.25, within the range of values for native protein structures of similar size.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    The C57BL/6 mouse AHR has commonly served as the reference AHR in studies reporting AHR affinity values.

    “Among these AHRs, the C57BL/6 mouse mAHR has been commonly used as the reference AHR for comparison purposes in many studies reporting AHR affinity values from one or several species.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Many fish species have multiple distinct AHR genes and proteins that differ in TCDD affinity.

    “Molecular analysis of several fish species revealed that many contain multiple distinct AHR genes and gene products that vary in their affinity for TCDD (60).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Hahn M. E., 2006, J. Exp. Zool., Part A source

  • May combine two claims.

    Interpretationown data

    These two amino acids are important for TCDD binding and TCDD-dependent DNA binding by zfAHR1a.

    “These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All mutant zfAHR1a constructs were fully sequenced.

    “All mutant zfAHR1a constructs were fully sequenced.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    PROCHECK validation showed good stereochemical quality for all models, with 85-94% of residues in the most favored Ramachandran areas and overall G-factors from -0.15 to -0.05.

    “PROCHECK validation indicated a good stereochemical quality for all the models, with 85 - 94% of residues belonging to the most favored areas of the Ramachandran plot and the overall G-factors ranging from -0.15 to -0.05 (this index ranges from -0.5 to 0.3 for structures solved at 1.5 Å resolution).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    AHRs with TCDD affinity 3- to 10-fold lower than the C57BL/6 mouse AHR are classified as medium affinity, including DBA mouse and human AHRs.

    “Those AHRs with TCDD binding affinity that was consistently 3- to 10-fold lower than that of C57BL6 mAHR were designated as having ‘medium’ binding affinity AHRs (Table 1) and included DBA mice and human AHRs (53,55,58,59).”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [53] Ema M., 1994, J. Biol. Chem. source
    • cites [55] Jensen B. A., 2001, Toxicol. Sci. source
    • cites [58] Poland A., 1994, Mol. Pharmacol. source
    • cites [59] Denison M. S., 1985, Comp. Biochem. Physiol., Part C: Pharmacol., Toxicol. Endocrinol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Killifish have at least two distinct AHRs: kfAHR1a with low TCDD affinity and kfAHR2a with medium TCDD affinity.

    “For example, killifish contain at least two distinct AHRs, with kfAHR1a having low affinity for TCDD (61) and kfAHR2a having medium TCDD affinity (unpublished results).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] Hahn M. E., 2004, Pharmacogenetics source

  • Classified as probably not a substantial claim.

    Premiseown data

    AHRs with TCDD affinity equal to or greater than that of the C57BL/6 mouse AHR are classified as high affinity, including rat, hamster, rabbit, guinea pig, beluga whale, harbor seal, and chicken AHRs.

    “Those AHRs with an affinity for TCDD equal to or greater than that of the C57BL/6 mAHR were classified as having ‘high’ TCDD binding affinity; these included rat, hamster, rabbit, guinea pig, beluga whale, harbor seal and chicken AHRs (Table 1).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites (Table 1)

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

  • May combine two claims.

    Interpretationown data

    The mutagenesis data confirm the zebrafish LBD model predictions and experimentally show that residues 296 and 386 are important for TCDD binding.

    “Overall, the mutagenesis data confirm the predictions made from comparative analysis of LBD models of the zebrafish AHRs and demonstrate experimentally the importance of amino acids at positions 296 and 386 in facilitating TCDD binding.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Optimal electrostatic interactions with TCDD for high-affinity binding may require a polar residue such as Ser in the region of the faced pair Ser359-Ala375 in mAHR or Ala364-Ser380 in chAHR.

    “It is possible that optimal electrostatic interactions with the TCDD molecule needed for high affinity binding require the presence of a polar residue, like Ser, in the region defined by the faced residue pair Ser359-Ala375, in mAHR, or Ala364-Ser380 in chAHR.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Interpretationown data

    Although Y296H or T386A partially restored ligand binding (~50%), the ligand-dependent change needed for AHR transformation and ARNT dimerization did not occur.

    “Overall, these results suggest that even though partial restoration of ligand binding occurs (~50%) with zfAHR1a containing the Y296H or T386A mutations, the ligand-dependent change in the AHR necessary to stimulate AHR transformation and ARNT dimerization fails to occur.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    The increased binding of zfAHR1a with Y296H+T386A may stem from greater stability of the AHR LBD and/or the AHR protein complex.

    “While the underlying mechanism of the increased ligand binding of zfAHR1a containing the Y296H+T386A mutation is unclear, it is possible that the enhanced binding may result from increased stability of the AHR LBD and/or AHR protein complex.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Interpretationreasoning

    Different inter-residue interactions in the central cavity may further restrict ligand access, since Tyr296, Thr386, and His388 would create a different electrostatic field and the facing hydroxyls of Tyr296 and Thr386 may form a hydrogen-bond network.

    “Moreover, the presence of different inter-residue interactions in the central part of the cavity may further limit the access of ligand as the Tyr296, Thr386, His388 residues would create a different electrostatic field, and the two facing hydroxyl groups of Tyr296 and Thr386 may generate a network of hydrogen bonds among the side chains.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationreasoning

    Greater hsp90 binding or stability within the LBD could stabilize AHR, reduce inactivation of ligand binding, and increase total [3H]TCDD binding.

    “one can envision that increased binding and/or stability of hsp90 within the LBD could further stabilize the AHR, leading to decreased inactivation of AHR ligand binding activity and consequently an increase in the overall amount of [3H]TCDD specific binding.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    TCDD binding in the chAHR LBD may be stabilized by specific interactions with amino acids at or near these positions.

    “it is conceivable that TCDD binding in the chAHR LBD may be stabilized by specific interactions with amino acids in or adjacent to these positions.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Interpretationreasoning

    The same steric feature appears in the modeled cavities of kfAHR1a and kfAHR2a and may explain their lower binding relative to zfAHR2.

    “This steric characteristic is also observed in the modeled cavities of the other two fish AHRs with low or medium TCDD binding affinity, kfAHR1a and kfAHR2a, and it may explain the reduced binding of these AHRs with respect to the zfAHR2.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The homology models accurately represent the native three-dimensional structures of the AHR LBDs.
    • Rests on an assumption: The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

  • May combine two claims.

    Interpretationown data

    The dimerization and DNA-binding interfaces of zfAHR1a appear functional.

    “These results also demonstrate that the dimerization and DNA binding interfaces of zfAHR1a appear functional”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Interpretationown data

    Mutations that fully restore TCDD binding also restore TCDD-dependent transformation and DNA binding.

    “Overall, the site-directed mutagenesis results reveal that the mutations that can fully restore ligand (TCDD) binding activity can also restore TCDD-dependent transformation and DNA binding.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro synthesized AHR proteins fold and function like native AHR proteins.
    • Rests on an assumption: The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

  • May combine two claims.

    Interpretationcitation

    These findings indicate that the zfAHR1a transactivation domain is functional and that its activation occurs by a ligand-selective mechanism distinct from zfAHR2.

    “These results not only indicate that the transactivation domain of zfAHR1a is functional, but that its activation appears to occur by a ligand-selective mechanism that is distinct from that of the zfAHR2 transactivation domain.”
    Quote in the paperSubstantial claim
58 of 58

  • The paper relies on this without support.

    The largest internal cavity identified by CASTp in each AHR LBD model corresponds to the ligand-binding site.

    “It ranks the cavities by size, where the largest one is usually the binding site.”

  • The paper relies on this without support.

    The homology models accurately represent the native three-dimensional structures of the AHR LBDs.

    “based on our homology model, are expected to increase the internal cavity volume and restore the electrostatic environment of the LBD.”

  • The paper relies on this without support.

    The CASTp-identified cavities correspond to the actual ligand-binding pockets of the AHR LBDs.

    “The analysis of structural pockets and cavities, performed by the CASTp server indicates the presence of a buried cavity in the core of each modeled domain.”

  • The paper relies on this without support.

    The in vitro synthesized AHR proteins fold and function like native AHR proteins.

    “Ligand ([3H]TCDD) specific binding to wild-type and mutant in vitro synthesized zfAHR1a proteins was performed using sucrose density centrifugation.”

  • The paper relies on this without support.

    The introduced mutations cause only the intended local cavity changes and no unintended global structural changes.

    “These specific amino acid substitutions change the specific zfAHR1a residues into those present in zfAHR2 and, based on our homology model, are expected to increase the internal cavity volume and restore the electrostatic environment of the LBD.”

  • The paper relies on this without support.

    The COS-7 luciferase reporter assay reflects the transactivation function of zfAHR1a in vivo.

    “we examined TCDD-dependent transcriptional activity of wild-type and mutant zfAHR1a in COS-7 cells transiently co-transfected with the AhR-responsive luciferase reporter plasmid pGudLuc6.1.”
6 of 6

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?

  • Previous comparative analysis of AhR LBD homology models of different species revealed that zfAhR1a has a dramatically shortened binding cavity compared to mouse AhR due to sidechains of three residues at the center of the cavity, which reduce available internal space to only the region at the entrance (Fα/Gβ site).

    “Our previous comparative analysis of the AhR LBDs homology models of different species revealed that zfAhR1a has a dramatically shortened binding cavity compared to the mouse AhR46 and this is due to the sidechains of three residues at the center of the cavity, which reduce the available internal space to only the region at the entrance of the cavity (Fα/Gβ site).”

    From Modeling the binding of diverse ligands within the Ah receptor ligand binding domain (2019) · cites this paper as [46]

    The Results and Discussion section (“Fish AHR LBD models”; page unavailable) attributes zfAHR1a’s dramatically shortened modeled cavity to steric hindrance from three side chains. But that analysis compares zfAHR1a with zebrafish AHR2, not mouse AhR, and the cited passage does not locate the remaining space specifically at the Fα/Gβ entrance. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Mutation of these residues in zfAhR1a to those present in mouse AhR restored the zfAhR1a ability to bind TCDD.

    “A confirmation of the role of this reduction on differential binding was provided by the evidence that mutation of these residues in zfAhR1a to those present in mouse AhR restored the zfAhR1a ability to bind TCDD46.”

    From Modeling the binding of diverse ligands within the Ah receptor ligand binding domain (2019) · cites this paper as [46]

    The Results and Discussion section reports that the Y296H/T386A double mutation restored TCDD binding in zfAHR1a. It identifies the substituted residues as those present in zebrafish AHR2, not mouse AhR; that part of the citing sentence is not supported. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. Denison M. S., 1998, Molecular Biology Approaches to Toxicology, 393
  2. 2010, The PyMOL Molecular Graphics System
  3. Poland A., 1994, Mol. Pharmacol., 46, 915
  4. Karchner S. I., 2011, Toxicol. Sci., 110, 79

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Authors
Fraccalvieri, D., Soshilov, A. A., Karchner, S. I., Franks, D. G., Pandini, A., Bonati, L., Hahn, M. E., & Denison, M. S.
Affiliations
  • University of Milano-Bicocca (IT)
  • University of California, Davis (US)
  • Woods Hole Oceanographic Institution (US)
  • King's College London (GB)
Corresponding author
Denison, Michael S.
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2013-01-14
Volume
52
Issue
4
Pages
714-725
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi301457f
PMC
PMC3568667
OpenAlex
W2167510551
Cited by
65
References
63
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 4 · 2020: 7 · 2019: 11 · 2018: 5 · 2017: 5 · 2016: 8 · 2015: 7 · 2014: 4 · 2013: 4
FWCI
5.63
Type
journal article
Language
en
Open access
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • BOA (University of Milano-Bicocca) (submittedVersion)
  • Brunel University Research Archive (BURA) (Brunel University London) (submittedVersion)
  • Research Portal (King's College London) (submittedVersion)
Funders
National Institute of Environmental Health Sciences (P42ES004699, R01ES007685, R01ES006272, P42ES007381)
Topics
  • Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Toxic Organic Pollutants Impact
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
Keywords
aryl hydrocarbon receptor, ligand-binding domain, homology model, 2,3,7,8-tetrachlorodibenzo-p-dioxin, ligand-binding cavity, species differences, site-directed mutagenesis, structure-function relationship, transcription factor activation
MeSH terms
Polychlorinated Dibenzodioxins, Amino Acid Sequence, Animals, Binding Sites, Chlorocebus aethiops, Environmental Pollutants, Humans, Ligands, Models, Molecular, Molecular Sequence Data, Protein Binding, Transcriptional Activation, Mutagenesis, Site-Directed, Protein Structure, Tertiary, Receptors, Aryl Hydrocarbon, COS Cells, Amino Acid Substitution, Amino Acid Motifs, Zebrafish Proteins, Avian Proteins, Structural Homology, Protein

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.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-09-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-09-28.

Cited sources are checked by gpt-6-sol.

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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