Biopolymers (Peptide Science), 2016 July; 106(4), 547-554. doi: 10.1002/bip.22795 

α-helix-peptides composing the human nuclear receptor ERRγ competitively provoke inhibition of functional homomeric dimerization

Full text link




Bisphenol A receptor ERRγ physiologically functions as homodimer 

Xiaohui Liu1, Hirokazu Nishimura1, Akina Fujiyama1, Ayami Matsushima1, Miki Shimohigashi2, Yasuyuki Shimohigashi1,*

1Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Science, and The Research-Education Centre of Risk Science, Kyushu University, Fukuoka 819-0395, Japan

2Division of Biology, Department of Earth System of Science, Faculty of Science, Fukuoka University, Fukuoka 814-0180, Japan

*Correspondence should be addressed to Yasuyuki Shimohigashi, E-mail:



Estrogen-related receptor g (ERRγ) is a constitutively active nuclear receptor functioning as a transcription factor. Recently, we demonstrated that bisphenol A (BPA), one of the nastiest endocrine disruptors, binds strongly to ERRγ. In the DNA transcription, ERRγ binds to a single half site designated as ERRE that has only a single binding motif. However, in regard to the subunit structure, it remains a matter of controversy whether ERRγ functions as a monomer or dimer. Because the ligand binding domain (LBD) of ERRγ was in a homodimer form in its X-ray crystal structure, the peptide fragments present in the dimer interfaces would perturb or destabilize the dimer structure by inhibiting the mutual interaction among ERRγ molecules. Thus, in order to demonstrate the essential functional homodimer structure of ERRγ, we utilized the peptides corresponding to the α-helix peptides no. 7 (H7), H9, and H10/11 in order to test such inhibitor activity. These selections were done based on structural analysis of the X-ray crystal structures of ERRγ-LBD, which forms a head-to-head dimer structure. Peptides were evaluated by means of a luciferase reporter gene assay, in which ERRγ exhibited a high constitutive activity with no ligand. When the peptide was expressed in the HeLa cells together with ERRγ, these peptides clearly showed a concentration-dependent activity inhibition, indicating that ERRγ is indeed homodimerized as required for DNA transcription activity. Although α-helix peptides were observed only in the cytoplasm when expressed solely, each peptide showed a distinct translocation from the cytoplasm to cell nucleus when expressed together with ERRγ. Such a nuclear translocation of peptides was due to the binding of peptide to ERRγ molecule consisting of the nuclear localization signal. The present results indicated that ERRγ functions as a genuine homodimer with symmetrical dimeric interface regions in LBD.

PMID: 26662629

Keywords: bisphenol A; constitutive activity; dimerization; ERRγ; estrogen-related receptors; nuclear receptors.


Additional text

During gene expression, transcription factors are essential to initiate the transcription of DNA to mRNA. Nuclear receptors (NRs) are a family of such transcription factors that function in the cellular nucleus. NRs play central roles in the regulation of cell growth and differentiation, lipid metabolism, cholesterol homeostasis, and many processes. NRs are known to be ligand-dependent. However, about one-fourth of NRs are constitutively active, being self-activated with no ligands. For instance, estrogen-related receptor g (ERRγ) is almost fully active without ligand binding and its apo form in an activation conformation has been demonstrated by X-ray crystallography (Figure 1). The endocrine disruptor bisphenol A (BPA) affects various genes and hormones even at merely physiological levels at very low doses. The risk of exposure to BPA and its endocrine-disrupting activities has been a cause of concern, especially for fetuses, infants, and children, who are particularly vulnerable to the adverse effects of chemicals [1]. We demonstrated that BPA binds extremely strongly to ERRγ, but retaining its high constitutive self-activating transcription activity and also its activation conformation (Figure 1) [2-4]. ERRγ is an orphan receptor particularly rich in both the fetal brain and the placenta, and also is a probable candidate for involvement in prostatic growth and development [5, 6].



Figure 1. The X-ray crystallographic homodimer structure of ligand-binding domain of estrogen-related receptor g (ERRγ-LBD) with and without bisphenol A. (A) Head-to-head or face-to-face overall structure of the ERRγ-LBD homodimer with BPA (red color) in the ligand-binding pocket (PDB accession code: 2E2R). One ERRγ-LBD molecule (green color) is coupled with another ERRγ-LBD molecule in brown color. (B) ERRγ-LBD homodimer with no ligand (PDB accession code: 1KV6). Also, one ERRγ-LBD molecule (green color) is coupled with another ERRγ-LBD molecule in brown color. (C) Inhibitory interaction between ERRγ-LBD and FLAG-tagged α-helix H10/11-peptide, in which H10/11 in the parent ERRγ molecule (green color) complexes interacts with ERRγ-H10/11-peptide (red), in stead of another molecule of ERRγ. This displacement is expected to prevent a homodimerization of ERRγ molecules, resulting in a loss of transcription activity.


ERRγ binds to classic estrogen response elements (EREs) and to extended half-site core sequences named ERR response elements (ERREs) [7]. However, although ERRγ has been frequently studied, the question of whether it functions in a monomer or homodimer form remains a controversial one. Because there are no reports addressing this question on a molecular basis, the bivalency of ERRγ remains a pressing issue. In the X-ray crystal structure, ERRγ-ligand-binding domain (LBD) is in a homodimer form (Figure 1) [4]. The dimer interface is formed as a series of amino acid residues present in α-helix 10 (H10) of ERRγ-LBD. Meanwhile, we identified a lysine cluster constructed with four Lys residues (Lys354, Lys355, Lys357, and Lys360) present in H7, and Lys355 and Lys357 were found to be essential for homodimerization of ERRγ. It should be noted that the counterpart residues are present in H9 of another ERRγ molecule.


On the basis of these structural backgrounds, we hypothesized that this firm homodimerization must be essential for ERRγ’s transcriptional activity, and thus we intended to examine whether ERRγ functions as a homodimer. Our strategy in the present study was to perturb the protein fold, or construction, of the dimeric structure of ERRγ by using peptides present at the dimer interface.


Design of ERRγ-LBD α-helix peptides for dimerization inhibition

We reasoned that, if a peptide involved in a dimer formation co-existed with ERRγ, that peptide would compete with ERRγ to interact with the target ERRγ molecule. We therefore selected three different α-helix peptides, H7, H9, and H10/11, for interaction at the dimerization interface. Thus, we prepared these selected α-helix peptides, which were N-terminally elongated with a FLAG-tag peptide for their immunocytochemical identification. Amino acid sequences of these FLAG-tagged peptide α-helix peptides are as follows: i.e., ERRγ-H7-peptide, MDYKDDDDKGLLDLNNAILQLVKKYKSMKLE; ERRγ-H9-peptide, MDYKDDDDKNSDSMH- IEDVEAVQKLQDVLHEALQDYEAGQHMEDPR, and ERRγ-H10/11-peptide, MDYKDDDDKMEDPRRAGKMLMTLPLLRQTSTKAVQHFYNIKLEGKVPM- HK. We also prepared α-helix peptide H10/11 of the androgen receptor AR-LBD because of its high distinct dissimilarity to ERRγ-LBD-H10/11.


Inhibitory activity of α-helix peptides in the transcription assay of ERRγ

Our method in the present study was to perturb the protein-protein interaction, or to prevent the construction of the dimeric structure of ERRγ, by using α-helix peptides in the dimer interfaces. ERRγ is highly constitutively active with no ligand, and this activity appears to be due to the activation conformation in a homodimerization. If homodimerization was perturbed or prevented, ERRγ’s transcription activity would be abolished. Thus, we selected a reporter gene assay to test the ability of the α-helix peptides of ERRγ to inhibit the transcription activity of ERRγ. The inhibition activity of ERRγ-LBD α-helix peptides in the HeLa cells was assessed by luciferase transfection assay. The activity was measured in the presence of the respective ERRγ-LBD α-helix peptides, the expression plasmids of which were co-transfected together with the pcDNA3.1(+)/ERRγ-full plasmid (0.6 mg), pGL3/3x ERRE plasmid (0.9 mg), and 10 ng/dish of pSEAP plasmid as an internal control in the medium. The amount of plasmid DNA necessary for each transfection was 0.001-1.0 μg/dish net weight.


When all the α-helix peptides, ERRγ-H7-peptide, ERRγ-H9-peptide, and ERRγ-H10/11-peptide, were co-transfected, respectively, with full-length ERRγ, ERRγ’s high constitutive activity was decreased in a dose-dependent manner. These α-helix peptides were equally active to inhibit the basal transcription activity of ERRγ (Figure 2). When 1.0 mg/dish was transfected, all the α-helix peptides showed a definitive inhibition (89-90%) of original ERRγ’s activity. When 0.1 mg/dish was transfected, ERRγ-H10/11-peptide was most active with approximately 50% inhibition. ERRγ-H7-peptide and ERRγ-H9-peptide showed approximately 10% and 40% inhibition, respectively (Figure 2). The fact that α-helix peptides of ERRγ-LBD inhibit the basal constitutive activity of ERRγ clearly indicate that α-helix peptides bother homodimerization of ERRγ, which is crucial for ERRγ to function as a nuclear transcription activation factor.



Figure 2. Inhibitory activity of ERRγ-LBD α-helix peptides for the basal constitutive activity of full-length ERRγ in the luciferase reporter gene assay in the HeLa cells. Bar graphs show the potency (%) relative to the basal constitutive activity of the full-length ERRγ (100%). α-helix ERRγ-H7, ERRγ-H9, and ERRγ-H10/11-peptides were tested for their ability to inhibit ERRγ. The negative control experiments were carried out with AR-LBD α-helix peptide H10/11 (AR-H10/11-peptide). The net weights per dish of plasmid DNA necessary for each transfection were 0.01 μg (A), 0.1 μg (B), and 1.0 μg (C). An internal control that distinguishes the transcriptional level from co-transfected α-helix peptides in transfection efficiency was achieved by co-transfecting a second plasmid that constitutively expresses an activity that can be clearly differentiated from SEAP. The assays were performed at least three times (P<0.0001).


Localization of FLAG-tagged α-helix peptides

The FLAG-epitope peptide attached to the N-terminal of α-helix peptides can be used for the identification of subcellular localization patterns of the inhibitory peptide. Nuclear receptors move into the cell nucleus, because their protein molecules consist of the nuclear localization signal. The FLAG-tagged α-helix peptides are unable to move into the cell nucleus by themselves, but those would move into the nucleus upon forming a complex with ERRγ. Otherwise, it would stay in the cytoplasm. Thus, the localization of the FLAG-tagged peptide in the cell nucleus would substantiate its interaction with the ERRγ molecule and the inhibitory activity.


Figure 3 shows the anti-FLAG immunolabeling of α-helix ERRγ-H10/11 peptide in the HeLa cells. The peptide was expressed at its highest concentration for the full inhibition of ERRγ. When the peptide was solely expressed, it was observed only in the cytoplasm as shown in Figure 3A. No immunolabeling was detected in the cell nucleus. In contrast, when the H10/11 peptide was co-expressed with ERRγ, immunolabeling (green) was detected in the cell nucleus as well as the cytoplasm (Figure 3B). Some of cells showed green immunolabeling mainly in the cell nucleus (Figure 3C). These results clearly imply that the peptide was transferred from the cytoplasm to the nucleus by means of the ERRγ molecule.



Figure 3. Localization of FLAG-tagged α-helix ERRγ-H10/11 peptide under the gene co-expression with or without a full-length ERRγ in the HeLa cells. (Panel A) FLAG-tagged ERRγ-H10/11 peptide was solely expressed in the cells. (Panels B and C) FLAG-tagged ERRγ-H10/11 peptide was co-expressed with a full-length ERRγ. FLAG peptide was detected by using rabbit polyclonal anti-FLAG(TM) antibody.



When green immunolabeling was detected only in the cell nucleus (Figure 4A), the anti-ERRγ immunolabeling was also performed simultaneously, detecting Cy3 red immunolabeling of ERRγ in the cell nucleus (Figure 4B). As a result, the entirely merged images were confirmed as shown in Figure 4C, revealing that ERRγ and the H10/11 peptide were completely co-localized and interacted tightly. Similar results were also obtained for the two other α-helix peptides, H7 and H9 (data not shown).




Figure 4. Localization of FLAG-tagged α-helix ERRγ-H10/11 peptide and full-length ERRγ under their gene co-expression in the HeLa cells. Evaluation of localization was carried out by co-staining for FLAG-tagged H10/11-peptide and ERRγ, to which rabbit anti-FLAG(TM) polyclonal antibody (pAb) and mouse anti-ERRγ monoclonal antibody (mAb) were used, respectively. The cells were observed by using FITC-conjugated goat anti-rabbit IgG antibody for FLAG-tagged H10/11-peptide (panel A), and Cy3 conjugate Goat anti-mouse IgG (H+L) secondary antibody for ERRγ (panel B). Both immunolabeling were finally merged together (panel C).


Inhibitory mechanism of α-helix peptides for ERRγ-LBD dimerization

The full-length ERRγ molecule forms a homodimer structure only at the LBD, and thus its LBD behaves as a simple competitive binding system. The expressed H10/11-peptide would interact with H10 in ERRγ, resulting in the formation of an ERRγ-H10/11-peptide complex (Figure 1C). This could prevent the homodimerization between the two ERRγ molecules, resulting in the displacement of one of two ERRγ molecules by the H10/11-peptide. This displacement is direct evidence that the homodimerization is in a bulk chemical equilibrium, and the α-helix peptide is able to perturb this equilibrium by competing for the dimerization interface area. Clearly, this interference produces a physiologically inactive ERRγ-α-helix peptide complex. The present study conclusively demonstrated that ERRγ indeed functions as a homodimer.



  1. Viberg, H., Lee, I. (2012) A single exposure to bisphenol A alters the levels of important neuroproteins in adult male and female mice. 33, 1390-1395. doi: 10.1016/j.neuro.2012.09.002
  2. Takayanagi,, Tokunaga, T., Liu, X., Okada, H., Matsushima, A., and Shimohigashi, Y. (2006) Endocrine disruptor bisphenol A strongly binds to human estrogen-related receptor gamma (ERRgamma) with high constitutive activity. Toxicol. Lett. 167, 95-105. doi: 10.1016/j.toxlet.2006.08.012
  3. Okada, H., Tokunaga, T., Liu, X., Takayanagi, S., Matsushima, A., and Shimohigashi, Y. (2008) Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor g (ERRγ). Health Perspect. 116, 32-38. doi: 10.1289/ehp.10587
  4. Matsushima, A., Kakuta, Y., Teramoto, T., Koshiba, T., Liu, X., Okada, H., Tokunaga, T., Kawabata, S., Kimura, M., and Shimohigashi, Y. (2007) Structural evidence for endocrine disruptor bisphenol A binding to human nuclear receptor ERRγ. Biochem. 142, 517-524. doi: 10.1093/jb/mvm158
  5. Takeda, Y., Liu, X., Sumiyoshi, M., Matsushima, A., Shimohigashi, M., and Shimohigashi, Y. (2009) Placenta expressing the greatest quantity of bisphenol A receptor ERRγ among the human reproductive tissues: Predominant expression of type-1 ERRγ J. Biochem. 146, 113-122. doi: 10.1093/jb/mvp049
  6. Poidatz, D., Dos, Santos E., Brulé, A., De, Mazancourt P., Dieudonné, M.N. (2012) Estrogen-related receptor gamma modulates energy metabolism target genes in human trophoblast. Placenta 33, 688-695. doi: 10.1016/j.placenta.2012.06.002
  7. Razzaque, M.A., Masuda, N., Maeda, Y., Endo, Y., Tsukamoto, T., and Osumi, T. (2004) Estrogen receptor-related receptor gamma has an exceptionally broad specificity of DNA sequence recognition. Gene 340, 275-282. doi:10.1016/j.gene.2004.07.010