International Journal of Molecular Sciences

Article Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

Samantha C. Faber 1, Anatoly A. Soshilov 1, Sara Giani Tagliabue 2 , Laura Bonati 2 and Michael S. Denison 1,*

1 Department of Environmental Toxicology, University of California, Davis, CA 95616, USA; [email protected] (S.C.F.); [email protected] (A.A.S.) 2 Department of Earth and Environmental Sciences, University of Milano-Bicocca, Milan 20126, Italy; [email protected] (S.G.T.); [email protected] (L.B.) * Correspondence: [email protected]; Tel.: +1-(530)-752-3879



Received: 7 August 2018; Accepted: 6 September 2018; Published: 10 September 2018


Abstract: The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

Keywords: Ah receptor; AhR; indirubin; TCDD; in vitro; in silico

1. Introduction The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) ligand-dependent transcription factor that mediates the induction/repression of a large battery of genes involved in diverse signaling pathways [1,2]. While localized in the cytosol, the AhR is held stably in an open ligand-binding conformation by its association with a complex of co-chaperone proteins, including two molecules of hsp90 (heat shock protein 90kDa), XAP2 (HBV X-associated protein 2, also called the AhR-interacting protein (AIP)), and p23 (phosphoprotein 23kDa) [3–6]. Binding of ligand within the cavity of the AhR ligand binding domain (LBD) triggers conformational changes in the AhR structure that exposes a nuclear localization sequence and stimulates nuclear translocation of the liganded AhR protein complex. Within the nucleus, the dimerization of AhR with the Ah receptor nuclear translocator (ARNT) protein causes dissociation of the AhR-associated

Int. J. Mol. Sci. 2018, 19, 2692; doi:10.3390/ijms19092692 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 2692 2 of 18 proteins and conversion of the AhR into its high-affinity DNA binding form [7]. This process is referred to as AhR transformation, and it effectively enables the ligand: AhR: ARNT complex to bind to its specific DNA binding site, the dioxin responsive element (DRE), adjacent to target genes and mediate AhR-dependent gene expression [8,9]. Recent studies have also reported that the AhR can heterodimerize with other nuclear proteins (KLF6 and RelB) and stimulate gene expression via distinctly different DNA binding sites [10–12]. Similar to the ligand-activated pregnane X receptor (PXR), a member of the steroid, thyroid and retinoic acid receptor superfamily, the AhR is promiscuous in its propensity to bind structurally diverse compounds, and significant differences in ligand binding specificity and ligand-specific AhR functionality have been reported across species [13]. The promiscuity reported for PXR ligands has been shown to result from the ability of these structurally diverse ligands to differentially bind to residues within the PXR ligand binding cavity [14,15]. By analogy, differences in the binding of ligands within the AhR LBD could also account for the observed structural diversity of AhR ligands, and this possibility is supported by several recent reports [16–18]. Examination of polymorphisms and mutations within the murine AhR (mAhR) LBD has identified several residues that contribute to some of the observed differences in affinity and selectivity of AhR ligands [17–19]. For example, while the ability of TCDD and related high-affinity halogenated and polycyclic aromatic hydrocarbon (HAHs and PAHs, respectively) ligands to activate the mAhR is significantly reduced by mutation of histidine 285 to tyrosine (H285Y), the structurally divergent ligand YH439 still activates the AhR [18]. Soshilov and Denison [17] not only identified a distinct region of the mouse AhR LBD (inclusive of amino acids H285, F289, F318, and H320) that was responsible for ligand-specific activation of the AhR, but mutations of isoleucine 319 (I319) into various other amino acids revealed significant diversity in AhR ligand selectivity. These results are consistent with differential interactions of structurally diverse agonist ligands with residues within the AhR ligand binding cavity. Interestingly, mutation of phenylalanine 318 (F318) revealed a role for this residue in differentiating between agonist and antagonist modes of action of a chemical [17]. Identification of antagonists that are species-specific [20,21] and/or ligand-selective [22] is also consistent with differential binding of chemicals within the AhR LBD, which can allow for ligand-selective activation or inhibition of the AhR [10]. In-depth analyses of the human AhR (hAhR) have demonstrated that it exhibits distinct differences in ligand-binding affinities, transformation efficiency, gene expression, and downstream biological and toxic effects when compared to rodent AhRs [23,24]. Although the hAhR was once considered an impaired AhR isoform due to reduced ligand binding and differential gene induction following activation by classical AhR ligands (2,3,7,8-tetrachlorodibenoz-p-dioxin (TCDD, dioxin) and polycyclic aromatic hydrocarbons), detailed molecular analysis revealed that a single amino acid difference (position 375 in the mAhR) between the “high-affinity” AhR (alanine in the AhRb allele) and “low-affinity” AhR (valine in the mAhRd allele and present in the hAhR) was responsible [25,26]. This was confirmed when mutation of A375 in the high-affinity mouse AhR to valine (A375V) resulted in a ~10-fold lower affinity and activation potency of the mAhR by TCDD [27,28], making the mAhR more similar to that of the hAhR. Significant species differences in AhR ligand-binding specificity and potency have also been reported more recently [23,24,26,29,30]. For example, while the AhR agonist indirubin (IR) has been shown to have a 10-fold greater potency as an inducer of AhR-dependent gene expression than TCDD in human hepatoma cells, it is ~10-fold less potent than TCDD in mouse hepatoma cells [23]. Additionally, IR was shown to be a more efficacious activator than TCDD of the hAhR in vitro and in cells in culture. For ligand-specific differences in hAhR response to manifest in a single cell type, significant differences in the binding interactions between ligands and amino acids within the hAhR LBD must occur, and this must translate into alterations in hAhR and/or hAhR: hARNT structure/function that ultimately lead to distinct ligand-dependent differences in AhR response. The documented ability of the AhR antagonist CH223191 to inhibit TCDD- but not IR-dependent gene expression in rat hepatoma cells suggests that these two ligands differentially interact with the AhR LBD [22]. However, the specific interactions and mechanisms responsible for, Int. J. Mol. Sci. 2018, 19, 2692 3 of 18 or contributing to, the enhanced potency and response of the hAhR to IR remains to be elucidated. We hypothesize that the enhanced potency of IR as an agonist of the hAhR, compared to the mAhR, results from species-specific differences in the interaction of IR with residues within the human and mouse AhR LBDs, but the specific interactions remain to be identified. Here we describe the results of studies using a combination of site-directed mutagenesis and AhR functional analysis to examine the mechanism(s) responsible for differences in the ability of IR to bind to and activate the hAhR and mAhR.Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 18

2. Resultsbe elucidated. We hypothesize that the enhanced potency of IR as an agonist of the hAhR, compared to the mAhR, results from species-specific differences in the interaction of IR with residues within 2.1. Comparisonthe human of the and Relative mouse PotencyAhR LBDs, and but Efficacy the specific of AhR interactions Agonists remain in Human to be identified. and Mouse Here Cells we describe the results of studies using a combination of site-directed mutagenesis and AhR functional IR hasanalysis been to reported examine the to mechanism(s) preferentially responsible stimulate for differences the hAhR in the [23 ability,29,31 of– IR33 to]; bind however, to and whether this phenomenonactivate the is hAhR specific and mAhR. for IR or whether other AhR ligands show similar species differences remains to2. beResults determined. In initial experiments, we examined the ability of several structurally diverse chemicals (Figure1) to activate AhR-dependent gene expression in mouse and human cells. TCDD, TCDF,2.1. Comparison BNF and of 3-MCthe Relative induced Potency luciferase and Efficacy reporterof AhR Agon geneists in expression Human and Mouse in human Cells HG2L6.1c1 cells in a concentration-dependentIR has been reported to manner preferentially (Figure stimulate2), although the hAhR maximal[23,29,31–33]; induction however, whether was not this observed with BNFphenomenon or 3-MCin is specific these for cells. IR or Notwhether only other do Ah theseR ligands results show similar reveal species a similar differences rank remains order potency to be determined. In initial experiments, we examined the ability of several structurally diverse for these compoundschemicals (Figure in 1) both to activate mouse AhR-dependent and human gene cells, expression but the in mouse relative and human inducing cells. potency TCDD, of each compoundTCDF, was BNF ~10-fold and 3-MC less induced in the luciferase human HG2L6.1c1reporter gene expression cells (Supplemental in human HG2L6.1c1 Table S1).cells in Interestingly, a distinct speciesconcentration-dependent difference in the manner rank (Figure order 2), potency although ofmaximal reporter induction gene was induction not observed were with observed by IR, ITE andBNF FICZ. or 3-MC In mousein these H1L6.1c3cells. Not only cells, do these these resu compoundslts reveal a similar stimulated rank order reporter potencygene for these induction to compounds in both mouse and human cells, but the relative inducing potency of each compound maximal levelswas ~10-fold comparable less in the to human that with HG2L6.1c1 TCDD cells and (Supplemental they were between Table S1). 50–250-foldInterestingly, lessdistinct potent than TCDD. Inspecies contrast, difference in human in the rank HG2L6.1c1 order potency cells, of reporter IR was gene ~7-fold induction more were potent observed than by IR, TCDD, ITE and FICZ was equipotentFICZ. to TCDD In mouse and H1L6.1c3 ITE was cells, ~100-fold these compounds less potent stimulated thanTCDD reporter (Figure gene induction2; Supplemental to maximal Table S1). Additionally,levels these comparable three to compounds that with TCDD induced and they were reporter between gene 50–250-fold expression less potent in human than TCDD. cells In to a level contrast, in human HG2L6.1c1 cells, IR was ~7-fold more potent than TCDD, FICZ was equipotent to significantlyTCDD greater and ITE than was that~100-fold obtained less potent with than TCDD TCDD alone;(Figure 2; a Supplemental phenomenon Table known S1). Additionally, as ‘superinduction’ of AhR-dependentthese three genecompounds expression induced relative reporter togene TCDD. expression While in human the exact cells mechanism(s) to a level significantly responsible for the observedgreater superinduction than that obtained response with TCDD is not alone; known, a phenomenon we have known previously as ‘superinduction’ described of several AhR- possible mechanismsdependent that may gene contributeexpression relative to synergistic to TCDD. Wh increasesile the exact in AhR-dependentmechanism(s) responsible gene expressionfor the [34]. observed superinduction response is not known, we have previously described several possible These resultsmechanisms are not that only may consistent contribute to with synergistic differences increases in in the AhR-dependent ability of IR, gene ITE expression and FICZ [34]. to bind to the humanThese and results mouse are AhRs,not only butconsistent they with demonstrate differences in that the ofability the of tested IR, ITE compounds, and FICZ to bind IR to exhibited the the greatest species-specifichuman and mouse differences AhRs, but inthey response. demonstrate Accordingly, that of the tested further compounds, studies focusedIR exhibited on investigationthe of speciesgreatest differences species-specific in IR-dependent differences activation in response. of the Accordingly, hAhR and mAhR.further studies focused on investigation of species differences in IR-dependent activation of the hAhR and mAhR.

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Figure 1. FigureStructures 1. Structures of the of AhR the AhR agonists agonists examined examined for for species species specificity. specificity. (A) Halogenated (A) Halogenated aromatic aromatic hydrocarbons (HAHs); (B) Polycyclic aromatic hydrocarbons (PAHs) and PAH-like; (C) indole- hydrocarbons (HAHs); (B) Polycyclic aromatic hydrocarbons (PAHs) and PAH-like; (C) indole-containing containing compounds. compounds. Int. J. Mol. Sci. 2018, 19, 2692 4 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 18

350 150 HG2L6.1c.1 A TCDD HG2L6.1c.1 TCDD 300 IR 125 TCDF

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0 0 -14-12-10-8-6-4 -14 -12 -10 -8 -6 -4 Chemical Concentration [M] Chemical Concentration [M] FigureFigure 2. 2. IRIR preferentially preferentially induces induces reporter reporter gene gene tran transcriptionscription in in HG26.1c1 HG26.1c1 cells. cells. AhR-dependent AhR-dependent gene gene transcriptiontranscription was was determined determined in in ( (AA)) human human HG2L6.1c.1 HG2L6.1c.1 and and ( (BB)) mouse mouse H1L6.1c.3 H1L6.1c.3 hepatoma hepatoma cell cell lines lines thatthat contain contain a a stably stably transfected transfected AhR-responsive AhR-responsive lu luciferaseciferase reporter reporter gene. gene. Cells Cells were were incubated incubated with with DMSODMSO (1%, (1%, vv//vv),), TCDD TCDD (0.1–100 (0.1–100 nM), nM), TCDF TCDF (0.1–100 (0.1–100 nM), nM), BNF BNF (0.001–10 (0.001–10 µM),µM), 3MC 3MC (0.001–10 (0.001–10 µM),µM), IRIR (0.001–10 (0.001–10 µM),µM), ITE (0.001–10 µM),µM), and FICZ (0. (0.001–10001–10 µM)µM) for 4 h. Luciferase activity (Relative LightLight Units Units (RLUs)) (RLUs)) was was normalized normalized to to the the maximal maximal induction induction observed observed with with TCDD TCDD in in each each cell cell line. line. Values represent the mean ± SDSD of of nine nine independent independent analyses. analyses. 2.2. The Human AhR Ligand Binding Domain Is Essential for Ligand-Selective Activation by IR 2.2. The Human AhR Ligand Binding Domain Is Essential for Ligand-Selective Activation by IR Ligand-selective and species-specific differences in AhR activation suggest that the diversity in Ligand-selective and species-specific differences in AhR activation suggest that the diversity in response may result from differential binding of structurally diverse ligands within the binding cavity response may result from differential binding of structurally diverse ligands within the binding of the AhR LBD [1,17,18,22,28]. To examine this, we first compared the ability of IR and TCDD to cavity of the AhR LBD [1,17,18,22,28]. To examine this, we first compared the ability of IR and TCDD stimulate in vitro transformation and DNA binding of the mAhR and hAhR by gel retardation analysis. to stimulate in vitro transformation and DNA binding of the mAhR and hAhR by gel retardation These results not only revealed that IR can stimulate transformation and DNA binding of both in vitro analysis. These results not only revealed that IR can stimulate transformation and DNA binding of synthesized mouse and human AhRs (Figure3), but that IR was a more potent and efficacious activator both in vitro synthesized mouse and human AhRs (Figure 3), but that IR was a more potent and than TCDD of the hAhR, similar to the gene expression results (Figure2). To confirm that the increased efficacious activator than TCDD of the hAhR, similar to the gene expression results (Figure 2). To responsiveness of the hAhR was due to its LBD and not to other regions of the AhR, we examined confirm that the increased responsiveness of the hAhR was due to its LBD and not to other regions the ability of TCDD and IR to stimulate DNA binding of a chimeric mAhR (mAhR-hAhRLBD) in of the AhR, we examined the ability of TCDD and IR to stimulate DNA binding of a chimeric mAhR which the mAhR LBD has been replaced with the corresponding region of the hAhR LBD (Figure4A). (mAhR-hAhRLBD) in which the mAhR LBD has been replaced with the corresponding region of the These analyses revealed that the hAhR LBD was essential for the significantly enhanced DNA binding hAhR LBD (Figure 4A). These analyses revealed that the hAhR LBD was essential for the significantly signal observed with IR (Figure3). While the relative potency of TCDD was similar between the hAhR enhanced DNA binding signal observed with IR (Figure 3). While the relative potency of TCDD was and mAhR-hAhRLBD chimera, the lower degree of TCDD-stimulated DNA binding compared to similar between the hAhR and mAhR-hAhRLBD chimera, the lower degree of TCDD-stimulated IR suggests that differences in the efficacy of each compound as AhR activators may account for the DNA binding compared to IR suggests that differences in the efficacy of each compound as AhR diminished reporter gene transcription reported in human cells [23]. Additionally, although the relative activators may account for the diminished reporter gene transcription reported in human cells [23]. Additionally, although the relative potency of IR in the chimeric AhR was not as dramatically

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Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 18 potency of IR in the chimeric AhR was not as dramatically different from that of TCDD, as observed differentwith the hAhR,from that it was of stillTCDD, more as potent.observed These with results the hAhR, suggest it thatwas thestill hAhR more LBD potent. plays These an essential results suggestrole in ligand-selectivethat the hAhR LBD activation plays an by essential IR. However, role in ligand-selective since the mAhR-hAhRLBD activation by didIR. However, not completely since therecapitulate mAhR-hAhRLBD the potency did differencenot completely in IR recapitulate activation of the the potency hAhR, difference it is conceivable in IR activation that additional of the hAhR,regions it of is the conceivable hAhR may that play additional a role and/or regions that protein-proteinof the hAhR may interactions play a role of theand/or hAhR that with protein- ARNT proteinor other interactions partners may of contributethe hAhR with to enhancing ARNT or IR-dependent other partners hAhR may transformation contribute to enhancing efficiency andIR- dependentDNA binding. hAhR transformation efficiency and DNA binding.

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FigureFigure 3. 3. TheThe human human AhR AhR ligand ligand binding binding domain domain plays plays a major a major role role in ligand-selective in ligand-selective activation activation by IR.by In IR. vitroIn vitro synthesizedsynthesized wild-type wild-type or chimeric or chimeric AhRs AhRs and and ARNT ARNT were were incubated incubated in the in thepresence presence of solventof solvent control control DMSO DMSO (1%, (1%, v/v)v or/v AhR) or AhRagonists agonists TCDD TCDD (0.001–100 (0.001–100 nM) or nM) IR or(0.001–10,000 IR (0.001–10,000 nM) for nM) 2 hfor and 2 hanalyzed and analyzed by gel by retardation gel retardation assay. assay. (A) The (A )amount The amount of inducible of inducible protein-DNA protein-DNA complex complex at each at chemicaleach chemical concentration concentration was wasquantitated quantitated and andvalues values normalized normalized to tothe the amount amount of of complex complex formed formed B withwith a a maximal maximal activating activating concentration concentration of of TCDD TCDD (20 (20 nM). nM). ( (B)) Representative Representative gels gels are are shown. shown. Values Values represent the mean ± SD of nine individual replicate analyses. The arrows indicate the ligand-induced represent the mean ± SD of nine individual replicate analyses. The arrows indicate the ligand-induced protein-DNA complexes. protein-DNA complexes.

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C

FigureFigure 4. Comparison 4. Comparison of theof the AhR AhR PASB PASB ligand ligand bindingbinding domain of of the the human human and and mouse mouse AhR. AhR. (A) (A) StructuralStructural domains domains of of the the hAhR hAhR (850 (850 aa), aa), mAhR mAhR (805 (805 aa) aa) and and chimeric chimeric mAhR-hAhRLBD mAhR-hAhRLBD (811(811 aa)aa) proteins;proteins; (B) Sequence (B) Sequence alignment alignment of the of the mAhR mAhR and and hAhR hAhR PASB PASB LBDs LBDs with with non-consensus non-consensus residues residues indicatedindicated by asterisks by asterisks (red (red asterisk asterisk for thefor the internal intern differental different residue). residue). Coloring Coloring scheme scheme for for residues:residues: red, acidic;red, acidic; blue, blue, basic; basic; purple, purple, polar; polar; yellow, yellow, Cys; Cys; brown, brown, aromatic; aromatic; green, green, hydrophobic; hydrophobic; orange, orange, Ser,Ser, Thr; gray,Thr; Pro;gray, white, Pro; white, Gly. Secondary Gly. Secondary structure structure elements elements according according to DSSP to forDSSP the for modeled the modeled AhR LBDs AhR are reportedLBDs are above reported the sequencesabove the se (helices:quences light(helices: gray; lightβ-strands: gray; β-strands: dark gray) dark and gray) labeled and labeled with thewith PAS the structurePAS nomenclature;structure nomenclature; (C) Cartoon (C representation) Cartoon representation of the AhR of LBD the homologyAhR LBD modelshomology (structural models superposition(structural of: superposition mAhR, in gray,of: mAhR, hAhR, in gray, in orange). hAhR, in Nonconserved orange). Nonconserved residues residues are shown are asshown sticks. as sticks. Secondary structure elements are labeled with the PAS structure nomenclature. Secondary structure elements are labeled with the PAS structure nomenclature.

2.3. Point2.3. Point Mutations Mutations in the in mAhRthe mAhR LBD LBD Produce Produce a hAhR-Like a hAhR-Like Response Response with with IR IR GivenGiven the highlythe highly conserved conserved nature nature of theof the AhR AhR across across species species and and the the role role of of the the hAhR hAhR LBDLBD inin ligand-selectiveligand-selective activation activation by IR, by it isIR, likely it is that likely amino that acid amino differences acid differences within the within hAhR arethe responsible hAhR are for theresponsible enhanced for activation the enhanced by IR.activation Sequence by IR. analysis Sequence of theanalysis LBDs of ofthe the LBDs mouse of the and mouse human and human AhRs identifiedAhRs 13identified nonconserved 13 nonconserved residues (Figureresidues4B,C). (Figure Among 4B, C). these, Among only these, the only mAhR the A375 mAhR (valine A375 (valine in the in the hAhR) has the side-chain directly pointing into the AhR binding cavity previously determined hAhR) has the side-chain directly pointing into the AhR binding cavity previously determined by by homology modeling (Figure 4C) and confirmed by mutational analysis [27,28]. To determine homology modeling (Figure4C) and confirmed by mutational analysis [ 27,28]. To determine which which of the nonconserved residues play a role in the enhanced response of the hAhR to IR, a series of the nonconserved residues play a role in the enhanced response of the hAhR to IR, a series of of mutations within the mAhR LBD were generated, wherein residues in the mAhR were mutated to mutations within the mAhR LBD were generated, wherein residues in the mAhR were mutated to

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Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 18 the corresponding hAhR LBD residues. The relative ability of increasing concentrations of IR and the corresponding hAhR LBD residues. The relative ability of increasing concentrations of IR and TCDD to stimulate transformation and DNA binding of the mutant mAhRs was determined by gel TCDD to stimulate transformation and DNA binding of the mutant mAhRs was determined by gel retardation analysis. retardation analysis. These experiments identified two mAhR to hAhR mutations (H326Y and A349T) that enhanced These experiments identified two mAhR to hAhR mutations (H326Y and A349T) that enhanced the efficacy of IR-dependent DNA binding of the mAhR by IR, and one mutation (A375V) that the efficacy of IR-dependent DNA binding of the mAhR by IR, and one mutation (A375V) that increasedincreased the the relative relative potency, potency, but but not efficacy,not efficacy, of IR, of such IR, such that IRthat was IR nowwas equipotentnow equipotent to that to ofthat TCDD of (FigureTCDD5). (Figure The remaining 5). The remaining ten mutations ten mutations did not significantly did not significantly alter IR activation alter IR activation from that from of wild-type that of mAhRwild-type (Supplemental mAhR (Supplemental Figure S1. MutationFigure S1. Mutation of histidine of histidine 326 to tyrosine 326 to tyrosine (H326Y) (H326Y) and alanine and alanine 349 to threonine349 to threonine (A349T) (A349T) enhanced enhanced the ability the (efficacy)ability (efficacy) of IR toof stimulateIR to stimulate transformation transformation of the of the mAhR mAhR into its high-affinityinto its high-affinity DNA binding DNA binding form and form increased and increased the relative the relative potency potency of IR forof IR the for mAhR the mAhR to greater to thangreater that of than TCDD. that Theof TCDD. A349T The mutation A349T alsomutation resulted also in resulted the greatest in the increase greatest in increase the relative in the potency relative of IR aspotency compared of IR toas TCDDcompared (Figure to TCDD5) and (Figure this appeared 5) and this to appeared result primarily to result from primarily a significant from a significant decrease in thedecrease ability of in TCDD the ability to stimulate of TCDD AhR to stimulate DNA binding. AhR DNA Interestingly, binding. theInterestingly, A349T mAhR the A349T mutant mAhR almost entirelymutant recapitulated almost entirely the recapitulated enhanced response the enha ofnced hAhR response to IR (Figure of hAhR3). to IR (Figure 3).

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TCDD Indirubin DMSO DMSO B mAhR A375V A349T H326Y

FigureFigure 5. 5.Enhanced Enhancedsignaling signaling of of the the hAhR hAhR by by IR IRis associated is associated with withselected selected residues residues within the within hAhR the hAhRPASB PASB LBD. LBD. In Invitro vitro synthesizedsynthesized mutant mutant mAhRs mAhRs and and wild-type wild-type mARNT mARNT were were incubated incubated in inthe the presencepresence of DMSOof DMSO (1%, (1%,v/v v)/ orv) AhRor AhR agonists agonists in DMSOin DMSO (TCDD (TCDD (0.001–100 (0.001–100 nM) nM) or IRor (0.001–10,000IR (0.001–10,000 nM) fornM) 2 h, for followed 2 h, followed by analysis by analysis of AhR of DNAAhR DNA binding binding by gel by retardationgel retardation analysis. analysis. (A ()A The) The amount amount of inducibleof inducible protein-DNA protein-DNA complex complex at eachat each TCDD TCDD or or IR IR concentration concentration waswas quantitated,quantitated, and and values values normalizednormalized to to the the amount amount of of complexcomplex formed with with a amaximal maximal activating activating concentration concentration of TCDD of TCDD (20 (20nM). nM). (B (B) )Representative Representative gels gels are areshown. shown. Values Values represen representt the mean the ± mean SD of ±nineSD individual of nine individual replicate replicateanalyses. analyses. The arrows The arrowsindicate indicate the ligand-induced the ligand-induced protein-DNA protein-DNA complexes. complexes.

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Consistent with these observations are the induction results obtained in COS-1 cells cotransfectedConsistent with with wild-type these observations or mutant are (H326Y, the induction A349T resultsand A375V) obtained mAhR in COS-1 expression cells cotransfectedvector and the withAhR-responsive wild-type or mutant luciferase (H326Y, reporter A349T plasmid and A375V) pGudLuc6.1. mAhR expression While TCDD vector was and comparable the AhR-responsive to IR as an luciferaseinducer reporterof luciferase plasmid activity pGudLuc6.1. in COS-1 While cell TCDDs cotransfected was comparable with to wild-type IR as an inducer mAhR, of luciferaseIR was a activitysignificantly in COS-1 more cells efficacious cotransfected inducer with than wild-type TCDD in mAhR, cells cotransfected IR was a significantly with the hAhR, more efficacious the mAhR- inducerhAhRLBD than and TCDD the in mutant cells cotransfected A349T mAhR with (Figure the hAhR, 6). Similar the mAhR-hAhRLBD to the DNA binding and the results, mutant while A349T the mAhRmaximal (Figure induction6). Similar observed to the with DNA TCDD binding in cells results, cotransfected while the maximalwith the inductionA349T mutant observed mAhR with was TCDDsignificantly in cells repressed cotransfected compared with the to A349Twild-type mutant mAhR mAhR, the maximal was significantly induction repressed response comparedobserved with to wild-typeIR was significantly mAhR, the maximal increased induction above that response of wild-type observed mAhR with and IR was of that significantly of TCDD. increased This decreased above thatresponse of wild-type likely mAhRresults andfrom of the that reduced of TCDD. ability This decreasedof TCDD to response stimulate likely transformation results from theand/or reduced DNA abilitybinding of TCDD of mAhR to stimulate containing transformation this substitution and/or (Figure DNA 5) binding. The relative of mAhR enhancement containing this of reporter substitution gene (Figureinduction5). The by relativeindirubin, enhancement compared ofto reporterTCDD, in gene these induction experiments by indirubin, was somewhat compared lower to TCDD, than its inrelative these experiments response in wasthe earlier somewhat studies lower (Figure than 3). its However, relative response this was in not the surprising earlier studies and likely (Figure results3). However,from a combination this was not surprisingof factors. andFirst, likely the experiment results froms ain combination Figure 3 not of only factors. utilized First, stably the experiments transfected inhuman Figure 3hepatoma not only utilizedcells containing stably transfected a wild-type human hAhR hepatoma (with numerous cells containing amino acid a wild-type differences hAhR from (withthe mAhR), numerous but amino cells were acid differencesincubated fromwith IR the for mAhR), only 4 but h before cells were measurement incubated of with luciferase IR for only activity. 4 h beforeIn contrast, measurement the experiments of luciferase presented activity. in In Figure contrast, 6 utilized the experiments COS-1 cells presented transiently in Figuretransfected6 utilized with a COS-1mAhR cells that transiently contained transfecteda single amino with acid a mAhR substitu thattion contained in the LBD a single basedamino on the acidhAhR substitution and induction in theof LBDluciferase based activity on the hAhRwas examined and induction after 18–20 of luciferase h, which activity allowed was the examinedcells more after time 18–20to metabolically h, which alloweddegrade the the cells IR. moreThe dramatic time to metabolically difference in degrade the induction the IR. response The dramatic of TCDD difference and IR in with the induction the A349T responsemAhR is of not TCDD only and consistent IR with with the A349T their respective mAhR is notabilities only consistentto stimulate with AhR their transformation respective abilities and DNA to stimulatebinding, AhRbut it transformation suggests that andthere DNA are binding,significant but differences it suggests in that how there these are specific significant ligands differences interact inwith how residues these specific within ligands the AhR interact ligand with binding residues cavity within and/or the AhR how ligand their binding binding cavity may and/ortranslate how into theirdifferences binding in may AhR translate transformation/DNA into differences binding. in AhR transformation/DNA binding.

400 TCDD Indirubin

† 300 *

†

200 †

†

100 * † *

* Luciferase ActivityLuciferase (Percent ofMaximal mAhRTCDD) 0 mAhR hAhR mAhR- H326Y A349T A375V hAhRLBD AhR Construct FigureFigure 6. 6.mAhR mAhR point point mutations mutations alter alter AhR-dependent AhR-dependent reporter reporter gene gene transcription transcription following following TCDD TCDD and IRand treatment. IR treatment. COS-1 cellsCOS-1 transiently cells transiently transfected transfected with wild-type with wild or mutant-type mAhRor mutant and mAhR DRE-containing and DRE- reportercontaining pGudLuc6.1 reporter pGudLuc6.1 were treated were with tr solventeated with control solvent DMSO control (0.1%, DMSOv/v (0.1%,), TCDD v/v), (10 TCDD nM), (10 or nM), IR (1orµM) IR for(1 µM) 18–20 for h. 18–20 Cells wereh. Cells lysed, were and lysed, lysates and were lysa analyzedtes were foranalyzed firefly luciferasefor firefly activity. luciferase Asterisks activity. indicateAsterisks the indicate values that the are values significantly that are significantly different from different the mAhR from TCDD the mAhR or IR, as TCDD indicated or IR, by as Two-Way indicated ANOVAby Two-Way with p ANOVA< 0.05 and with (†) indicatep < 0.05 IRand values (†) indicate are significantly IR values different are signi fromficantly TCDD diff withinerent from the givenTCDD constructwithin the by Student’sgiven constructt-test p by< 0.05.Student’s Luciferase t-test activityp < 0.05. (relative Luciferase light activity units; RLU)(relative was light measured units; andRLU) correctedwas measured for background and corrected (DMSO) for background and normalized (DMSO) to mAhR and normalized TCDD levels. to ValuesmAhR representTCDD levels. the meanValues ±representSD of nine the individual mean ± SD replicate of nineanalyses. individual replicate analyses.

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2.4. Ligand Binding Analysis Reveals That the A349T Mutation Differentially Affects TCDD and IR 2.4. Ligand Binding Analysis Reveals That the A349T Mutation Differentially Affects TCDD and IR While the above results demonstrate that the insertion of an A349T mutation in the mAhR LBD significantlyWhile the reduced above resultsthe ability demonstrate of TCDD that to thestimulate insertion AhR of antransformation/DNA A349T mutation in binding the mAhR and/or LBD AhR-dependentsignificantly reduced gene expression, the ability ofthis TCDD mutation to stimulate enhanced AhR the transformation/DNAactivity of IR. However, binding whether and/or these effectsAhR-dependent result from gene an expression,alteration in this the mutation ability of enhanced these chemicals the activity to bind of IR. to However, the AhR whether is unknown. these Accordingly,effects result competitive from an alteration [3H]TCDD in the ligand ability binding of these analysis chemicals was carried to bind out to to the determine AhR is unknown. whether theAccordingly, A349T mutation competitive alters [3 H]TCDDthe relative ligand ability binding of [3H]TCDD analysis wasor IR carried to bind out to to the determine mAhR. In whether these experimentalthe A349T mutation conditions, alters although the relative ~10% more ability [3H]TCDD of [3H]TCDD bound or to IR the to in bind vitro to synthesized the mAhR. wild-type In these mAhRexperimental than to conditions, the mAhR-hAhRLBD although ~10% chimera, more [3[3H]TCDDH]TCDD boundbinding to to the thein vitroA349Tsynthesized mutant mAhR wild-type was notmAhR significantly than to the different mAhR-hAhRLBD from that chimera, of the [wild-type3H]TCDD bindingmAhR, toindicating the A349T that mutant this mAhRhAhR-specific was not mutationsignificantly had different no significant from thateffect of on the overall wild-type [3H]TCDD mAhR, specific indicating binding. that this The hAhR-specific relative ability/affinity mutation ofhad IR no to significantbind to each effect of onthese overall AhRs [3 H]TCDDwas then specificdetermined binding. by concentration-dependent The relative ability/affinity [3H]TCDD of IR to competitivebind to each binding of these AhRsanalysis was (Figur thene determined 7). These results by concentration-dependent not only revealed that [3 H]TCDDIR could competitivebind to the mAhR-hAhRLBDbinding analysis (Figure chimera7). Thesewith an results apparent not only affinity revealed greater that than IR could that for bind the to mAhR the mAhR-hAhRLBD (IC50 values of 0.82chimera ± 0.28 with nM an and apparent 17.67 ±1.66 affinity nM, greater respectively), than that but for insertion the mAhR of (ICthe50 A349Tvalues mutation of 0.82 ± in0.28 the nM mAhR and had17.67 no± 1.66significant nM, respectively), effect on the but affinity insertion of binding of the A349T of IR mutation (IC50 value in theof 4.61 mAhR ± 1.87 had nM) no significant (Supplemental effect Tableon the S3). affinity Interestingly, of binding although of IR (IC the50 value A349T of substitu 4.61 ± 1.87tion nM)had (Supplementalno significant effect Table on S3). the Interestingly, binding of TCDDalthough or IR the to A349T the mAhR, substitution a significant had no reduction significant in effectthe ability on the ofbinding TCDD and of TCDD an increase or IR toin thethe mAhR,ability ofa significantIR to stimulate reduction AhR in transformation/DNA the ability of TCDD andbinding an increase and AhR-dependent in the ability ofgene IR to expression stimulate AhRwas observed.transformation/DNA While A349 bindingdoes not and appear AhR-dependent to be involved gene in expressionAhR ligand was binding, observed. or at Whileleast the A349 affinity does ofnot binding, appear tothe be increased involved inDNA AhR binding ligand binding,response or suggests at least thethat affinity it plays of binding,a role in theligand-stimulated increased DNA AhRbinding transformation/DNA response suggests thatbinding. it plays a role in ligand-stimulated AhR transformation/DNA binding.

150 mAhR A349T mAhR- hAhRLBD 125

100

* 75 * *

50 * * * * * * * 25 * * * * H]TCDDSpecific Binding (Percent of Maximal mAhR) 3 [ 0 M M M M M M M M M M M M M n n n n n n n n 0 1n 1 0 0 1n 1 0n 0nM 0nM 1 0nM0 0n . 10nM 0 . 10n0 0 .1 1 0 0 100nM 0 1 0 10 10 10 10 Indirubin [M] FigureFigure 7. A349TA349T mutation mutation has has no no significant significant effect effect on on th thee relative relative affinity affinity of of IR IR for for mAhR. mAhR. InIn vitro vitro synthesizedsynthesized mAhR, mAhR, mutant mutant AhR, AhR, or or mAhR-hAhRLBD mAhR-hAhRLBD chimeric chimeric protein protein was was incu incubatedbated in in the the presence presence ofof 2 2 nM nM [ [33H]TCDDH]TCDD and and indicated indicated concentrations concentrations of IR for 30 min min and [ 33H]TCDDH]TCDD bound bound to to the the protein protein fractionfraction was was measured measured by by the the hydroxyapatite hydroxyapatite assay. assay. The The unprogrammed unprogrammed TNT TNT lysate lysate was was used used as asa nonspecifica nonspecific binding binding control, control, and and specific specific binding binding was was calculated calculated as a asdifference a difference between between the total the totaland nonspecificand nonspecific reactions. reactions. Values Values represent represent the mean the mean ± SD± ofSD nine of nineindividual individual replicate replicate analyses. analyses. The asterisksThe asterisks indicate indicate the values the values that thatare significantly are significantly different different from from the theno-competitor no-competitor reaction, reaction, as indicatedas indicated by the by theTwo-Way Two-Way ANOVA ANOVA with with p < p0.05.< 0.05. The Therelative relative affinity affinity of IR of for IR the for mAhR, the mAhR, A439T A439T and mAhR-hAhRLBDand mAhR-hAhRLBD was 17.67 was 17.67 ± 1.66,± 4.611.66, ± 4.61 1.87± and1.87 0. and82 ± 0.820.28 ±nM,0.28 respectively, nM, respectively, as determined as determined using nonlinearusing nonlinear regression regression (three-parameter) (three-parameter) analysis analysis of the ofcompetitive the competitive binding binding results. results.

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2.5. Molecular Docking Predicts Differences in TCDD and IR Binding within the mAhR and hAhR LBDs 2.5. Molecular Docking Predicts Differences in TCDD and IR Binding within the mAhR and hAhR LBDs To analyze the hypothesis that IR interacts with residues within the AhR ligand binding cavity in a mannerTo analyze distinctly the hypothesis different that from IR interactsthat of TCDD, with residues the binding within geometries the AhR ligand of these binding ligands cavity in the in amAhR manner and distinctly hAhR differentLBDs were from predicted that of TCDD, by molecular the binding modeling geometries methods. of these The ligands LBD in thestructures mAhR andpreviously hAhR LBDsgenerated were by predicted homology by modeling molecular [35] modeling were used methods. for molecular The LBD docking, structures and previously the three generatedforms of IR by were homology submitted modeling to docking [35] weresimulati usedons for (see molecular Materials docking, and Methods and the section). three forms The ofmost IR wereenergy-favored submitted tocomplex docking was simulations obtained with (seeMaterials the IR trans and stereoisomer Methods section). for both The mAhR most and energy-favored hAhR. The complexdocking poses was obtained of TCDD with and the IR IRin the trans mAhR stereoisomer LBD (Fig forure both 8A) mAhRconfirm and that hAhR. IR binds The within docking the poses cavity of TCDDwith different and IR inarrangement the mAhR and LBD interactions (Figure8A) comp confirmared that to IRTCDD. binds While within TCDD the cavity occupies with the different central arrangementpart of the cavity and interactionsand the two compared chlorine toatoms TCDD. reac Whileh the TCDD most internal occupies hydrophobic the central part region of the [35], cavity IR andbinds the nearer two chlorineto the entrance atoms of reach the cavity. the most This internal causes hydrophobic the loss of some region stabilizing [35], IR interactions binds nearer that to theare entrancecharacteristic of the of cavity.TCDD Thisbinding causes with the residues loss of in some the stabilizingmost buried interactions and hydrophobic that are region characteristic within the of TCDDcavity but, binding at the with same residues time, init theallows most a buriedgain of and stabilizing hydrophobic contributions region within with theresidues cavity near but, to at the samecavity time, entrance it allows (Fα and a gain Gβ of). stabilizingWhile the naturally contributions occurring with residues mutation near of A375 to the (mAhR) cavity entrance to V381 (F(hAhR)α and Gcausesβ). While the displacement the naturally occurringof the TCDD mutation binding of A375pose (mAhR)toward the to V381 entrance (hAhR) of the causes hAhR the cavity displacement (35), it ofslightly the TCDD affects binding the IR pose towardby maintaining the entrance the sa ofme the placement hAhR cavity observed (35), it in slightly the mAhR affects cavity the IR with pose a bymodified maintaining orientation the same of the placement molecular observed plane (Figure in the 8B). mAhR Therefore, cavity with the IR a modified molecule orientation contacts some of the of molecularthe residues plane predicted (Figure for8B). the Therefore, mAhR pose the at IR the molecule cavity contactsentrance some as well of theas some residues unique predicted ones. Despite for the mAhRthe similar pose placement at the cavity of entrance TCDD and as well IR at as the some entran uniquece of ones. the DespitehAhR binding the similar cavity placement (Figure of 8B), TCDD the anddistinct IR at chemical the entrance characteristics of the hAhR of the binding two ligands cavity (Figuregenerate8B), different the distinct stabilizing chemical interactions characteristics with the of theinternal two ligandsresidues. generate different stabilizing interactions with the internal residues.

Figure 8. BindingBinding geometries geometries of of TCDD TCDD (light (light green green)) and and indirubin (marine blue) predicted by molecular docking to the AhR PASB LBD homology models. ( A) mAhR in grey and (B) hAhR in orange. The nonconserved internal residuesresidues A375/V381 are are shown shown as as sticks. sticks.

3. Discussion It is well established that the AhR can bind andand be activated by a wide range of structurally diverse compounds compounds and and that that significant significant species species differ differencesences exist exist in ligand in ligand specificity specificity [1,2,36,37]. [1,2,36 ,The37]. Theemergence emergence of endogenous of endogenous AhR ligands AhR that ligands are both that structurally are both structurally diverse and diversestimulate and physiological stimulate physiologicalsignaling pathways, signaling highlights pathways, the highlightsimportance the of importance understanding of understanding the mechanisms the mechanismsby which these by whichcompounds these selectively compounds activate selectively the human activate AhR the relati humanve to AhR classical relative rodent to classical models rodentfor the modelspurpose for of theimproving purpose risk of improvingmanagement risk stra managementtegies and strategiesdevelopment and of development AhR pathway-specific of AhR pathway-specific therapeutics. therapeutics.Endogenous AhR Endogenous activator, AhR indirubin, activator, has indirubin, been detec hasted been at concentrations detected at concentrations of 0.2 nM in human of 0.2 nM urine in humanand shown urine to and modulate shown toAhR-dependent modulate AhR-dependent microbiome microbiome homeostasis homeostasis and inflammatory and inflammatory signaling pathways [24,29–31]. Potent induction of hAhR-dependent gene transcription in transgenic mice,

Int. J. Mol. Sci. 2018, 19, 2692 11 of 18 signaling pathways [24,29–31]. Potent induction of hAhR-dependent gene transcription in transgenic mice, transfected mouse hepatocytes, and human hepatoma cells indicates that IR preferentially activates the human AhR [23]; however, the mechanism of ligand-selective activation and whether structural divergence within the ligand binding domain plays a role in regulation of hAhR functionality had not been thoroughly investigated. By analogy with mechanisms that have been reported for ligand-dependent gene expression by steroid hormone receptors, ligand- and species-specific differences in the activation of the AhR could alter AhR and ARNT protein structure and thus influence recruitment/interaction with coactivators, transformation efficiency, protein-protein interactions, gene expression, and downstream signaling events. The ability of IR to enhance transformation and DNA binding efficiency in the hAhR and mAhR-hAhRLBD chimeric construct in vitro, relative to the mAhR, confirmed the significance of the hAhR LBD on conferring IR-dependent ligand-selective activation of the AhR. Insertion of the hAhR LBD into the mAhR did not completely recapitulate the hAhR phenotype, suggesting that the full enhanced response to IR may require additional human-specific AhR residues, protein-protein interactions and/or protein conformational changes that only occur within the full length hAhR and not in the mAhR-hAhRLBD chimera. In fact, it has been previously shown that the mAhR LBD coordinates with the C-terminal end to effectively transform the receptor and enable enhancement/repression of target gene transcription [24]. Likewise, the effects of species-specific LXXLL coactivators, interdomain influence, or crosstalk with other nuclear proteins could modulate AhR-dependent responsiveness to a given ligand across species [38]. The premise that the structure and functional activity of the AhR is dependent on the specific ligand bound within the cavity is supported by gene expression studies, where diverse AhR ligands produce distinctly different magnitudes of induction of the same gene at equipotent concentrations and also induce a ligand-specific set of AhR-dependent gene products [1,39]. While the majority of the known selective activators of the hAhR (i.e., indoxyl sulfate, indole, tryptamine, and 2AI) are indole-containing compounds [30], other selective AhR activators have also been identified. Cinnabarinic acid (CA), which is structurally similar to TCDD and does not contain an indole group, demonstrates clear ligand-selective AhR-dependent activation of stannocalcin 2 (Stc2) gene expression [39,40]. Chromatin immunoprecipitation analysis of the Stc2 promoter region identified several DREs that were selectively bound by CA-activated AhR and induced CA- and AhR-dependent induction of Stc2, while classical ligands (TCDD, β-naphthoflavone, 3-methylcholanthrene) failed to stimulate AhR binding and Stc2 gene expression [39]. The selective activation of the AhR by these and other structurally diverse AhR ligands (agonists and antagonists) suggests alternate binding within the AhR ligand binding domain, which could potentially stimulate distinct AhR conformational changes and downstream biological effects [18,21]. Site-directed mutagenesis and functional analysis of a series of mutant mAhRs that contained a single amino acid substitution in its LBD that was swapped for the corresponding residue in the hAhR LBD allowed identification of 3 individual mutations inserted into the mAhR LBD (H326Y, A349T and A375V) that significantly increased the potency of IR relative to that of TCDD (Figure5 and Supplemental Table S2); the remaining individual mutations had little or no significant effect on the relative potency of TCDD and IR. While one mutation (A349T) significantly increased the relative potency of IR from ~6-fold less potent than TCDD to ~50-fold more potent than TCDD, the remaining two mutations (H326Y and A375V) increased the relative potency of IR such that it became equipotent to TCDD. It should be noted that while the H326Y substitution actually had a greater effect on the relative potency of IR (compared to TCDD) than the A375V mutation, because the latter mutation also resulted in a significant decrease the relative potency of TCDD for the mAhR. The reduction in the affinity of the AhR for TCDD with this specific mutation was previously reported [27,28]. Overall, these results, combined with previous site-directed mutagenesis analysis [17] demonstrate that significant differences in binding do exist between TCDD and IR and suggest that the selectivity and responsiveness to IR may depend on multiple amino acids within the LBD. Int. J. Mol. Sci. 2018, 19, 2692 12 of 18

So how might these specific residues within the hAhR contribute to the specificity and increased potency of IR compared to that of TCDD? Based on our homology models of the mAhR LBD [35], two of these residues (H326 (Y332 in the hAhR) and A349 (T355 in the hAhR)) are in distinctly different locations within the LBD. H326 is located in the Fα helix or long helical connector of the PASB (Figure4C), which in other PAS proteins plays an important signaling role and can be displaced from the β-sheet in different ligand-dependent ways to form a cavity suitable for a variety of ligands or cofactors [41,42]. Since the side chain of H326/Y332 points out of the binding cavity and toward the G-strand of the AhR β-sheet, mutation of this residue would not be expected to affect AhR ligand binding; however it could alter AhR functionality by altering ligand-selective transformation and DNA binding and thereby affecting the interaction of AhR with its partner proteins (e.g., hsp90 and/or ARNT). This hypothesis is supported by our recent models of the AhR:ARNT dimer in which the helical connector of the mAhR PASB is involved in the dimerization interface [43,44]. The homology model of the AhR LBD indicates that the A349 residue is located in a loop connecting the G and H strands of the β-sheet and similar to H326, its side chain does not point into the AhR ligand binding cavity (Figure4C). However, given the effect of its substitution on IR-dependent DNA binding, this residue must play a role in the enhanced ability of IR, compared to TCDD, to stimulate transformation/dimerization of the AhR with ARNT and conversion of the AhR into its DNA binding form. However, since our recent modeling of AhR: ARNT dimerization [43,44] indicates that this residue does not appear to be involved in the dimerization interface with ARNT, we infer that it plays a more significant role in interactions with other partners or with different regions of the AhR and ARNT proteins that enhance the ability of IR to stimulate AhR transformation/DNA binding. The presence of valine at position 381 in the hAhR LBD has been documented to significantly reduce the binding affinity of TCDD due to steric hindrance by its side chain pointing directly in the center of the modeled LBD cavity, likely disrupting the interaction of TCDD with a closely associated histidine residue [27,28]. Mutation of alanine in this position in the mAhR to valine decreases the binding affinity of TCDD [27,28]. Interestingly, while it was reported that mutation of this residue in the hAhR (V381A) or mAhR (A375V) had no effect on the binding of IR to the AhR [23], the gel retardation analysis results presented here (Figure5 and Supplemental Table S2) revealed that a A375V mutation in the mAhR increased the relative potency of IR in stimulating mAhR transformation and DNA binding ~5-fold. Together, these results are consistent with significant differences induced by in the binding of IR and TCDD within the cavities of these AhRs, with IR binding affinity apparently unaffected by mutation of this residue. While the exact mechanism(s) by which TCDD and IR bind to and activate the AhR remain to be elucidated, the results presented here demonstrate that the differential interactions of these ligands with residues within the LBD of the hAhR are responsible for the enhanced signaling potency of IR. One avenue in which to gain additional insights into similarities and differences in the interaction of TCDD and IR with residues inside the binding cavities of the hAhR and mAhR is by performing molecular docking of these ligands into the homology models of their LBDs [45]. Docking analysis of TCDD within both the AhR LBDs has been previously reported by our laboratory [35], and the docking simulations of IR binding presented here have allowed prediction of the binding geometries and direct comparison with those of TCDD. Despite the fact that IR maintains the characteristic planarity of TCDD and other dioxin-like AhR ligands, it shows different molecular dimensions and shape, higher polarity, and has functional groups that may form hydrogen bonds. As a consequence, our docking analysis revealed binding geometries of IR in the mAhR LBD distinctly different from that of TCDD (Figure8A). While TCDD showed stabilizing interactions with residues both in the middle and in the inner part of the cavity [35], the IR binding site was predicted to be closer to the entrance of the cavity and this resulted in different stabilizing interactions of IR with internal residues. Interestingly, docking to the hAhR LBD revealed that the V381 residue, known to adversely affect the binding of TCDD [27,28], perturbs both the TCDD and IR binding poses causing a similar placement of the two ligands within the cavity (Figure8B). TCDD is displaced toward the cavity entrance and loses many of the stabilizing interactions that occur in the mAhR cavity. In contrast, the V381 residue does not Int. J. Mol. Sci. 2018, 19, 2692 13 of 18 hinder the binding of IR, which occupies a region near to the entrance of the cavity like that in the mAhR, but only induces a rotation of the molecular plane. Taken together, these findings suggest that alanine or valine similarly affect the binding affinity of IR to the AhR in agreement with experimental evidence [23] and suggest that IR may have a higher relative binding affinity to the hAhR compared to TCDD. This prediction is consistent with the [3H]TCDD competitive binding results that suggest higher binding affinity of IR to the hAhR LBD. The differential binding poses of IR and TCDD within the cavity also likely contribute, along with other sequence and structural differences in the AhRs, to the subsequent transformation events responsible for the enhanced potency and efficacy of IR. Site-directed mutagenesis and structure-function analysis, including those based on homology models, have aided in providing clear evidence for differential binding of structurally diverse ligands within the mouse and human AhR ligand binding cavity. Although part of the AhR: ARNT dimer structure (bHLH-PASA) was experimentally determined recently [46,47], the lack of experimental 3D structures of both the AhR PASB LBD and the full length AhR: ARNT dimer including this domain still hampers a complete mechanistic understanding of binding by structurally diverse ligands and how ligand binding activates the AhR. Targeting of the AhR and/or AhR signaling pathway holds significant promise for development of therapeutic pharmaceuticals for treatment of a variety of human diseases. Thus, future studies identifying and characterizing the residues responsible for ligand-dependent AhR transformation, ARNT-dimerization, DNA binding and transcriptional activation will be crucial for increasing our understanding of the basic molecular mechanisms underlying the functional activity of the AhR and its diversity in biological and toxicological responses.

4. Materials and Methods

4.1. Chemicals TCDD was obtained from Dr. Stephen Safe (Texas A&M University), [3H]TCDD (14.3 Ci/mmol) was obtained from ChemSyn Laboratories (Lenexa, KS, USA), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) was from Accustandard (New Haven, CT, USA). [32P]-ATP (~6000 Ci/mmol) was from Perkin Elmer Life & Analytical Sciences. The structures of the specific AhR ligands used in these studies are shown in Figure1. 3-Methylcholanthrene (3MC), β-naphthoflavone (βNF) and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA), 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) and 6-formylindolo[3,2-b]carbazole (FICZ) were from Tocris Bioscience (Minneapolis, MN, USA) and IR from AmplaChem (Carmel, IN, USA). All chemical stocks and dilutions were prepared in DMSO.

4.2. Plasmids The mouse AhR and ARNT expression plasmids, mβAhR/pcDNA3 and mβArnt/pcDNA3, have been previously described [17]. The chimeric mouse/human mAhR/hAhR expression plasmid, mAhR-hAhRLBD, contains a full-length mAhR cDNA in which the mouse PASB LBD was replaced with the corresponding region of the hAhR LBD. Human AhR and ARNT were obtained from Christopher Bradfield (University of Wisconsin, Madison, WI, USA) and Oliver Hankinson (University of California, Los Angeles, CA, USA), respectively, and were inserted in pcDNA3β [26] to generate hβAhR/pcDNA3 and hβARNT/pcDNA3. Point mutations of mβAhR/pcDNA3 were designed using Agilent QuikChange Primer Design and Mus musculus aryl-hydrocarbon receptor transcript variant mRNA (NM_013464.4, nucleotides 367 to 2784). A list of mouse AhR mutagenic primers can be found in Table S4. Site-directed mutagenesis was carried out using the Agilent Technologies QuikChange Lightning Mutagenesis Kit and all constructs were verified by sequencing.

4.3. Hydroxyapatite [3H]TCDD Ligand Binding Assay [3H]TCDD specific binding to wild-type mAhR and the chimeric mouse/human mAhR-hAhRLBD synthesized in vitro using the Promega TNT Quick coupled transcription/translation rabbit reticulocyte lysate kit (Madison, WI, USA) was carried out as previously described [48]. [3H]TCDD Int. J. Mol. Sci. 2018, 19, 2692 14 of 18 specific binding was determined by subtracting the amount of [3H]TCDD bound to unprogrammed lysate (nonspecific binding) from the total amount of [3H]TCDD binding to lysate containing in vitro expressed AhR. The amount of [3H]TCDD specific binding remaining in the presence of competitor chemical was expressed as a percent of the total [3H]TCDD specific binding.

4.4. AhR DNA Binding (Gel Retardation) Assay Wild-type and mutant AhRs and ARNT were synthesized in vitro in the presence of unlabeled L-methionine, the resulting AhR and ARNT translation mixtures and MEDGK (25 mM MOPS (3-(N-morpholino) propanesulfonic acid; pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 150 mM KCl) were mixed in a 1:1:8 (v/v/v) ratio and incubated with DMSO (1% final concentration) or the indicated concentration of TCDD or IR for the indicated periods of time at room temperature. An aliquot of each incubation was mixed with [32P]-labeled double-stranded oligonucleotide containing the AhR-ARNT DRE3 DNA binding site (a dioxin responsive element from the upstream region of the murine Cyp1a1 gene [9] and protein-DNA complexes resolved by gel retardation analysis as previously described [48,49]. Gels were visualized using a FLA9000 Fujifilm Imager and protein-DNA complexes quantitated with Fujifilm MultiGauge software (FujiFilm Corporation, Valhalla, NY, USA).

4.5. Reporter Gene Assays Recombinant mouse (H1L6.1c3) and human (HG2L6.1c1) hepatoma cells containing a stably integrated AhR-responsive luciferase reporter gene plasmid (pGudLuc6.1) were plated (750,000 cells/well) in 96-well plates and incubated at 37 ◦C for 24 h prior to chemical treatment. Cells were incubated with DMSO (1% (v/v)) or the indicated concentration of test chemical in DMSO for 4 h, followed by visual inspection of the cells for toxicity, washing of the cells with phosphate-buffered saline (PBS), addition of 50 µL of passive lysis buffer (Promega) and lysis of cells for 20 min at room temperature with shaking [50]. Luciferase activity in each well was measured (integrating luminescence over 10 s with a 10 s delay) in an Orion microplate luminometer (Berthold Detection Systems, Bad Wildbad, Germany) following automatic injection of Promega stabilized luciferase reagent. Luciferase activity was corrected for background luciferase activity in DMSO-treated cells and values expressed as relative light units (RLU) or as a percent of the luciferase activity obtained with the maximally inducing concentration of TCDD.

4.6. Transient Transfection Assays COS-1 cells were plated at a density of 75,000 cells/well and allowed to attach overnight in a 24-well plate. Cells were transiently transfected (per well) with the following amounts per well: 2 µL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), 20 ng wild-type (mβAhR/pcDNA3) or mutant AhR expression plasmids (in pcDNA3.1), 20 ng of mβARNT/pcDNA3 or pcDNA3.1(+) and 200 ng pGudLuc6.1 [17]. Twenty-four hours after transfection, cells were incubated with DMSO (0.1%, v/v), TCDD (10 nM final concentration) or IR (1 µM final concentration) for 18 to 22 h, followed by washing with PBS, lysis and measurement of luciferase activity in 50 µL aliquots as described above.

4.7. Statistical Analysis Analysis of statistical significance of differences between experimental values was conducted using Two-Way ANOVA and EC50s and IC50s were calculated using nonlinear regression (three parameter) analysis in GraphPad Prism v.7 (La Jolla, CA, USA).

4.8. Molecular Modeling The mAhR and hAhR PASB LBD structures were generated previously by homology modeling starting from the following sequences: UniProt id P30561 and residues 278–384 for mAhR; UniProt id P35869 and residues 284–390 for hAhR. Three ligand-bound X-ray structures of the HIF-2α protein Int. J. Mol. Sci. 2018, 19, 2692 15 of 18

(PDB ID: 3F1O, 3H7W and 3H82) were used as templates [35]. Four out of the one hundred models with the best DOPE score obtained by MODELLER [51] for each species were selected as representatives of the conformational variety of the modeled LBD by cluster analysis. Each model was refined by Molecular Mechanics (MM) energy minimization using MacroModel [52] maintaining a template ligand inside the binding cavity, as previously described [35]. The IR molecular structure was downloaded from PubChem (CID: 5359405) and prepared with LigPrep [53]. Two stereoisomers may exist at pH = 7 (cis and trans); in addition, a deprotonated form of IR is also considered to be present in water solution; all three forms were submitted to MM energy minimization and to the subsequent docking protocol. Molecular docking was carried out using Glide extra precision (XP) [54,55]. An ensemble docking approach, based on ligand docking to the four representative conformations of the modeled receptor, was used to include receptor flexibility. To take into account the induced-fit effects, a further optimization of the docking poses was performed using MacroModel [52]. Finally, the binding free energy (∆Gbind) of the obtained ligand-receptor complexes was calculated with the MM Generalized Born Surface Area (MM-GBSA) method [56] implemented in Prime MM-GBSA [57], which includes evaluation of the solvation contributions by an implicit solvent model. The obtained ∆Gbind values allowed the selection of the best pose among the ensemble. Visualization of the models was accomplished using PyMOL [58].

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/19/9/2692/s1. Author Contributions: S.C.F., M.S.D., S.G.T. and L.B. contributed to the conception and design of the study, to the interpretation of the data and wrote the manuscript; S.C.F. and S.G.T. performed the experiments with assistance of A.S., S.G.T. and L.B. contributed to the modeling and docking studies, while S.C.F., M.S.D. and A.S. contributed to the biochemical and molecular analysis studies. Funding: This research was supported by the National Institute of Environmental Health Sciences (NIEHS) (R01ES007685), a NIEHS Superfund Research Grant (P42ES004699), and a Predoctoral NIEHS fellowship to SCF (T32ES007059). Conflicts of Interest: The authors declare no conflict of interest.

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