Fig 2: Mutations altering dimerization of Arnt and AhR PAS.A domains define a putative dimerization interface. (a) Residues found to be important for Arnt and AhR dimerization are illustrated on homology models of Arnt and AhR PAS.A domain. The mutations are coloured according to the severity of the defect in the lacZ reporter assay. For Arnt red: <60%, magenta: 61–70%, blue: 71–80% and green: >80% of wt activity (Figure 2a) and for AhR red: <60%, magenta: 61–80%, green: >80% of wt activity (Figure 5a). (b) Superimposition of PAS.A models of Arnt and AhR onto the structure 3F1P of HIF-2a/Arnt PAS.B (49) suggests dimerization occurs through different interfaces for PAS A and PAS B domains.

Fig 3: Arnt mutations differentially alter dimerization with other partner proteins. Activation of the CME driven reporter (pML-6c-wt) in 293T cells by wt or mutant HisMyc_Arnt together with hSim1_Myc (a), NPAS4_Myc (b) or hSim2s_Myc (c) shown as fold activation over basal levels (puro6). Data are mean ± SD of transfections performed in triplicate and are representative of two independent experiments. Hepa C4 cell lines expressing wt, E163K, S190P or D217G HisMyc_Arnt were treated with vehicle, YH439 or 2,2'-dipyridyl (DP) and assayed for CYP1A1 (d) or VEGF (e) mRNA induction by qRT-PCR. Data are mean ± SEM, n = 3. (f) Whole cell extracts from the Hepa C4 Arnt deficient and derived cell lines constitutively expressing wt, E163K, S190P or D217G HisMyc_Arnt were separated by SDS–PAGE, followed by western blotting for Myc tagged Arnt and a-tubulin loading control. Dashed line indicates removal of an empty lane from the image. (g) Activation of the E-box driven 4(CACGTG)TKMPluc reporter gene in 293T cells by wt or mutant HisMyc_Arnt. Data are mean ± SD of transfections performed in triplicate and are representative of two independent experiments.

Fig 4: Single amino acid mutations in Arnt PAS.A domain with disrupted AhR/Arnt heterodimerization isolated using RevB2H. (a) The strength of interaction between RNAPa_AhR278 and the potential ?CI_Arnt362 loss-of-interaction mutants selected as large blue colonies from RevB2H was measured in a standard forward bacterial two hybrid system with the lacZ reporter gene (28). Data are mean with 95% confidence intervals, shown as a percentage of ß-gal activity obtained with wt ?CI_Arnt362 protein, n = 9. bHLH: ?CI_Arnt bHLH domain only; *P < 0.05 (one tailed unpaired Student’s t-test). (b) Expression of mutant ?CI_Arnt362 relative to wt; mean ± SEM. from three independent bacterial extracts. Percent symbol denotes grouping by ß-gal activity relative to wt from (a). (c) Shows a representative western blot. (d) CD spectra of wt H6_Arnt362 and selected loss-of-interaction mutants, normalized to allow comparison with wt (30). CD spectra of all mutants are shown in Supplementary Figure S2. (e) The dimerization deficient mutations identified from RevB2H mapped onto the sequence of Arnt PAS.A, aligned with dPer PAS.A. dPer PAS.A is representative of PAS.A domains, which have one to three insert loops within the PAS domain fold (25). The positions of secondary-structure elements, including the predicted N-terminal a-helix are indicated above. The mutations are coloured according to the strength of disruption observed in the ß-gal assay; red: <60%, magenta: 61–70%, blue: 71–80% and green: >80% of wt activity, yellow: Hepa c4 mutation G341D (37).

Fig 5: Mutations in AhR PAS.A that alter dimerization with Arnt. (a) The strength of interaction between ?CI_Arnt362 and the potential RNAPa_AhR278 loss-of-interaction mutants selected as large blue colonies from RevB2H was measured in the forward bacterial two hybrid system with the lacZ reporter gene (28). Data are mean with 95% confidence intervals, shown as a percentage of ß-gal activity obtained with wt RNAPa_AhR278. bHLH: RNAPa_AhR bHLH domain only; *P < 0.05 (one tailed unpaired Student’s t-test). (b) AhR PAS.A mutations identified from RevB2H disrupted full-length AhR/Arnt function. Activation of the XRE-driven reporter gene in 293T cells by wt or mutant HisMyc_AhR and HisMyc_Arnt in response to vehicle or the ligand YH439, shown as fold activation over basal levels (puro6) from endogenous protein. Data are mean ± SD of transfections performed in triplicate and representative of two independent experiments. (c) Expression of wt and mutant HisMyc_AhR in the transfected cells used for the reporter gene assay in (b), detected by western blotting for Myc-tagged AhR and a-tubulin loading control. (d) AhR mutations deficient in dimerization with Arnt mapped onto the PAS.A sequence, illustrated with dPer PAS.A. See Figure 2e legend for details. Colouring of mutations according to the strength of disruption observed in the ß-gal assay (a): red: <60%, magenta: 61–80%, green: >80% of wt activity.