The crystal structure of the AhRR–ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription

2,3,7,8-Tetrachlorodibenzo-p-dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR–ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR–ARNT and AhR–ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR–ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR–ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription.

In the absence of ligands, AhR resides in the cytoplasm by forming a complex with heat shock protein 90 (14), X-associated protein 2 (15)(16)(17)(18), and p23 (19,20). Upon ligand binding, AhR translocates to the nucleus and forms a heterodimer with AhR nuclear translocator (ARNT), and this AhR-ARNT complex interacts with a specific DNA sequence-the xenobioticresponsive element (XRE)-to activate transcription (1, 2, 4 -7). However, the AhR-ARNT complex concurrently induces the expression of AhR repressor (AhRR), a negative-feedback regulator of AhR signaling that also interacts with ARNT and forms an AhRR-ARNT complex and down-regulates AhR signaling (21). Thus, elucidation of the mechanism of AhRRmediated transcriptional repression is critical for limiting excessive activation of AhR.
AhR, ARNT, and AhRR belong to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family of transcriptional regulators (22)(23)(24). Typically, bHLH-PAS family members form a heterodimer with other members of the same family through their N-terminal bHLH-PAS domains (Fig. 1A). Whereas the bHLH domain is responsible for DNA binding, the tandem PAS domains (PAS-A and PAS-B) are involved in protein-protein interaction and ligand binding (22)(23)(24). In AhR, ligand binding occurs at the PAS-B domain, but this ligand-binding domain is lacking in AhRR (21,23,24). The N-terminal bHLH-PAS region is well-conserved among bHLH-PAS family members, but the C-terminal region, which mediates transcriptional activation or repression through interaction with coactivator or corepressor molecules, is comparatively less conserved (22,24).
The mechanism of transcriptional repression by AhRR has been described in a few previous reports, but the mechanism remains debated (21,(25)(26)(27). Because AhR and AhRR are highly similar, AhRR, like AhR, heterodimerizes with ARNT and interacts with XRE DNA with high affinity. Therefore, AhRR competes with AhR for binding to ARNT and XRE and thus represses AhR signaling (21). Furthermore, SUMOylation of the C-terminal region of AhRR has been reported to be critical for recruiting various corepressor molecules, including ankyrin-repeat protein 2, histone deacetylase (HDAC) 4, and HDAC5, to the promoter region and thereby down-regulating transcription (25,27). Conversely, Evans et al. (26) have presented contrasting results and have argued that the competitive mechanism for ARNT and XRE binding and the corepressormediated mechanism cannot fully explain how AhRR represses transcription; the investigators have proposed the transrepression hypothesis (28), in which AhRR is considered to compete

Crystal structure of AhRR-ARNT heterodimer
with AhR for binding to unknown coregulatory proteins and promoter-bound transcription factors.
Here, to gain insights into the transcriptional repression mechanism of AhRR, we determined the crystal structure of the AhRR-ARNT complex, which exhibited a spatially distinct asymmetric domain arrangement among the bHLH-PAS family members. Our results provide key structural insights into the mechanisms by which AhRR represses transcription.
The final structural model contained one complex of AhRR-ARNT in the crystal asymmetric unit. The two bHLH and three PAS domains in the heterodimer were all defined in the electron-density map, although the electron density obtained for the PAS-A domain of ARNT was relatively poor (Fig. 1C). Overall, the AhRR-ARNT heterodimer exhibited an asymmetric intertwined domain organization featuring the shape of an inverted triangle, in which the PAS-A domain of AhRR was positioned at the center, the bHLH domains at the bottom, and the two PAS domains of ARNT at the two apexes at the top (Fig.  1D). The N-terminal regions and some of the flexible loops of both AhRR and ARNT were disordered. Each domain showed the canonical folds found in the bHLH-PAS family: the bHLH domains of both proteins exhibited a helix-loop-helix structure with two ␣-helices (␣1 and ␣3) connected by a linker region containing a short ␣-helix (␣2), and the three PAS domains (PAS-A of AhRR and PAS-A and PAS-B of ARNT) were composed of a central five-stranded ␤-sheet flanked by surrounding ␣-helices (Figs. 1B and 2). Each corresponding domain between AhRR and ARNT was similar, with root-mean-square deviation (RMSD) values of 0.7 and 2.0 Å for bHLH and PAS-A domains, respectively (Fig. 1B). The PAS-B domain of ARNT also resembled the PAS-A domains of AhRR and ARNT (RMSD ϭ 1.0 and 2.9 Å, respectively) (Fig. 1B).

Comparison between AhRR-ARNT and ARNT heterodimers formed with other bHLH-PAS family members
Among bHLH-PAS family members, AhRR is unique in that it lacks the PAS-B domain (21,(23)(24)(25)(26)(27). Therefore, we compared the structure of AhRR-ARNT with previously reported structures of bHLH-PAS family members. First, we compared the structure of AhRR-ARNT with that of AhR-ARNT encompassing only the bHLH and PAS-A domains, wherein human AhRR and human AhR share 55% sequence identity (30, 31) ( Figs. 2A and 4A). Because the residues of AhRR involved in the interaction with ARNT were mostly conserved in AhR ( Fig.  2A), the two complexes exhibited similar domain arrangements involving the conserved interfaces, with an RMSD of 1.6 Å (31) (Fig. 4A). The contact area of AhRR and ARNT (2,753 Å 2 ) in this region was comparable to that of AhR and ARNT (2,562 Å 2 ). Moreover, the AhR residues involved in the interaction with XRE DNA were perfectly conserved in AhRR (31) (Fig.  2A). Therefore, the AhRR-ARNT complex would bind to XRE DNA in a similar manner as the AhR-ARNT complex. The structural resemblance of this region provides the basis for the AhRR competition with AhR for interaction with ARNT and XRE DNA.
Next, we compared the AhRR-ARNT structure with the structures of heterodimers containing ARNT and other   (Fig. 4, A and B). This newly identified protein-protein interface between PAS-A (AhRR) and PAS-B (ARNT) contributed to the formation of the unique quaternary structure of AhRR-ARNT in the bHLH-PAS family. The unique interface between PAS-A (AhRR) and PAS-B (ARNT) was mainly formed by hydrophobic residues (Fig. 4C). The side chains of Trp-177, Ala-178, Met-179, Ile-199, Tyr-217, and Phe-220 of AhRR and those of Ile-364, Phe-375, Val-422, Phe-444, Phe-446, and Ile-458 of ARNT contributed to the hydrophobic interactions. Furthermore, hydrogen bonds formed between the following residues also contributed to the binding: Ala-178 (AhRR) and Arg-366 (ARNT), Arg-202 (AhRR) and Lys-419 (ARNT), Glu-216 (AhRR) and Val-422 (ARNT), and Tyr-217 (AhRR) and Gly-420 (ARNT) (Fig. 4C). Although the structures of the PAS-A domains of AhRR and AhR were similar, with an RMSD of 0.6 Å (Fig. 4D) (31), most of Figure 2. Sequence alignment of AhRR, AhR, and ARNT. A, sequence alignment of human AhRR and AhR. Secondary structure elements of AhRR are indicated above the alignment. Conserved residues are highlighted in red. AhRR residues interacting with ARNT in the AhRR-ARNT structure (this study) are indicated by blue asterisks (with bHLH-PAS-A) and green asterisks (with PAS-B). AhR residues interacting with ARNT and XRE in the AhR-ARNT-XRE structure (PDB code 5NJ8) (31) are indicated by yellow and black asterisks, respectively. Residues missing in the structural models are surrounded by gray boxes. B, sequence alignment of bovine, human, and mouse ARNT. Secondary structure elements of bovine ARNT are indicated above the alignment. Conserved residues are highlighted in red. ARNT residues interacting with AhRR (this study), AhR (in the AhR-ARNT-XRE structure, PDB code 5NJ8) (31), and HIF-2␣ (in the HIF-2␣-ARNT structure, PDB code 4ZP4) (29) are indicated by blue, yellow, and red asterisks, respectively. The bovine ARNT residues deleted for crystallization in this study are indicated by dashed lines (i)-(iv). Residues missing in the structural models are surrounded by gray boxes.

Crystal structure of AhRR-ARNT heterodimer
the residues in the PAS-A (AhRR)-PAS-B (ARNT) interface were not conserved between AhRR and AhR ( Figs. 2A and 4D). Notably, the ␣9 helix of PAS-A (AhRR) made a large contribution to the binding, whereas the corresponding region in AhR was disordered. These observations provide a structural basis for the specific interactions between PAS-A (AhRR) and PAS-B (ARNT), a characteristic feature of the AhRR-ARNT heterodimer.

Discussion
The most widely accepted model of AhRR-mediated repression of AhR signaling is based on the competitive mechanism, where AhRR competes against AhR for heterodimerization with ARNT and thus for binding to XRE DNA (Fig. 5A) (21). Supporting this model, the structure of AhRR-ARNT resembled that of AhR-ARNT in the bHLH-PAS-A region (Fig. 4A), and the residues involved in the interaction with ARNT were mostly conserved between AhRR and AhR ( Fig. 2A). Our results further indicated that AhRR-ARNT would efficiently compete with AhR-ARNT for DNA binding: although the structure of the AhRR-ARNT-XRE DNA complex is not available, we found that the AhR residues that interact with XRE DNA were perfectly conserved in AhRR ( Fig. 2A), and the bHLH domain was positioned similarly as in the AhR-ARNT complex (Fig. 4A). These findings validate the ability of the competition model to account for AhRR-mediated transcriptional repression to certain extent.
In contrast to the conservation of structural features between AhR-ARNT and AhRR-ARNT in the bHLH-PAS-A region, the PAS-B (ARNT) domain in AhRR-ARNT was located at a distinct position as in other complexes (Fig. 4A). Although the structure of the AhR-ARNT heterodimer across the entire   Crystal structure of AhRR-ARNT heterodimer domain) (33) is critical for the recruitment of corepressor molecules and the resultant transcriptional repression activity of AhRR-ARNT (Fig. 5B). AhRR and ARNT enhance the SUMOylation of each other through heterodimerization, whereas AhR does not enhance the SUMOylation of ARNT (25). In the transrepression hypothesis proposed by Evans et al. (26), which assumes the existence of unknown interaction partners common for AhR and AhRR, AhRR functions in a manner independent of its competition for ARNT and XRE or functions without its C-terminal region (Fig. 5C). Considering these findings together with the results of the structural analysis, it is tempting to speculate that the unique quaternary architecture identified in this study plays roles in recruiting SUMO E3 ligase for the SUMOylation of AhRR-ARNT or in interacting with the unknown molecules proposed in the transrepression model. However, further studies are required to test and validate this hypothesis.
In summary, we determined the crystal structure of the AhRR-ARNT complex, which revealed structural features of the heterodimer that are conserved and distinct among bHLH-PAS family members. Our findings advance the current understanding of the mechanism by which AhR activation is repressed by AhRR and thus should contribute to the development of a therapeutic strategy for limiting excessive activation of AhR.

Crystallization, data collection, and structure determination
The purified AhRR-ARNT complex was lysine-ethylated by using a Reductive Alkylation Kit (Hampton Research) according to the manufacturer's instructions and then purified using a Superdex 200 gel-filtration column. The protein solution used for crystallization contained ϳ10 mg/ml lysine-ethylated AhRR-ARNT in a buffer consisting of 10 mM Tris-HCl, pH 8.0, 350 mM NaCl, and 7% (v/v) glycerol. Crystals were grown at 293 K using the sitting-drop vapor-diffusion method; the crys-tallization droplets were prepared by mixing the protein solution (0.4 l), reservoir solution (0.4 l; 12.3% (w/v) PEG 20000 and 100 mM Hepes-NaOH, pH 7.3), and additive solution (0.1 l; 0.5 M dimethylethylammonium propane sulfonate).
The crystal structure of the AhRR-ARNT complex was solved through molecular replacement performed with Phaser (35) by using the coordinates of HIF-2␣-ARNT (Protein Data Bank (PDB) code 4ZP4) (29) and AhR (PDB code 4M4X) (36) as search models. The model was subject to iterative cycles of manual model building by using the program COOT (37) and restrained refinement by using REFMAC (38) ( Table 1). The quality of the refined model was evaluated using MolProbity (39), and the structural figures were prepared using CueMol. The coordinate and structure-factor data of the AhRR-ARNT complex have been deposited to Protein Data Bank under PDB code 5Y7Y.