Figure 1.
Characteristic 3D structural views of the bisphenol A-binding site in α-helix 7 (H7) of the ERRγ ligand-binding domain.
(A) Side view of the interaction, and (B) top view of the interaction between bisphenol A and amino acid residues. (Protein Data Bank with accession code: 2E2R [22]).
Table 1.
Receptor binding characteristics of [3H]BPA for wild-type ERRγ and its mutants in the saturation binding assay.
Figure 2.
The homologous competitive binding assays between [3H]bisphenol A and non-labeled bisphenol A for the wild-type ERRγ-LBD and its mutants.
The receptors used were the wild-type ERRγ and its mutant receptors. (A) Leu342-substituted ERRγ mutant receptors, (B) Leu345-substituted ERRγ mutant receptors, (C) Asn346-substituted ERRγ mutant receptors, and (D) Ile349-substituted ERRγ mutant receptors. The graphs show representative dose-dependent binding curves, which give the IC50 value closest to the mean IC50 from at least three independent experiments.
Table 2.
Receptor binding potency of BPA, 4-α-cumylphenol, and 4-OHT in the competitive binding assay using [3H]BPA for human nuclear receptor ERRγ and its mutants with site-directed mutagenesis in the BPA binding site amino acid residues.
Figure 3.
Structural interrelationships between the bisphenol A-binding site amino acid residues Leu342-Asn346/Leu345-Ile349 and their back support residues present in the binding pocket of ERRγ-LBD.
(A) Leu345 and its back support residue Ala431. There is no direct contact between Ala431 and BPA. (B) Ile349 and its back support residue Val313. There is no direct contact between Val313 and BPA. (C) Leu342 and its back support residue Leu268. (D) Leu268 and its back support residues Leu265 and Tyr330. This Leu268 binds directly to the phenol-A and B rings of BPA as a double-hook to connect both tightly. (E) Ile349/Asn346 and their simultaneous back support residue Leu324. (F) Four amino acids, Leu324, Tyr326, Tyr330, and Met332, stand on the pleated β-sheet, which makes a bottom plate of ERRγ-LBP. (G) Leu345 and its back support residue Lys430. (H) Leu342/Leu345 and their simultaneous back support residue His434.
Table 3.
Receptor binding potency of BPA, 4-α-cumylphenol, and 4-OHT in the competitive binding assay using [3H]BPA for human nuclear receptor ERRγ and its mutants with site-directed mutagenesis in the back support residues of BPA binding sites.
Figure 4.
The heterogeneous competitive binding assays between [3H]bisphenol A and 4-α-cumylphenol for the wild-type ERRγ-LBD and its mutants.
The receptors used were the wild-type ERRγ and its mutant receptors. (A) Leu345-substituted ERRγ mutant receptors, and (B) Asn346-substituted ERRγ mutant receptors. The graphs show representative dose-dependent binding curves, which give the IC50 value closest to the mean IC50 from at least three independent experiments.
Figure 5.
The results of luciferase-reporter gene assay to evaluate the biological activity of ERRγ and its Ala-substituted mutant receptors.
The percentage relative potencies of a series of mutant receptors were measured against the basal constitutive activity of the wild-type ERRγ receptor (100%). (A) Binding site residues, and (B) Back support residues. An internal control that distinguishes the transcriptional level from variations in transfection efficiency was achieved by co-transfecting a second plasmid that constitutively expresses an activity that can be clearly differentiated from SEAP. The mark “*” shows that no basal constitutive activity was observed for Ile310Ala and Val313Ala. The assays were performed in triplicate at least three times (P<0.0001).