br Experimental Procedures br Author Contributions
Introduction Estrogen-related receptors (ERRα, ERRβ, and ERRγ) are orphan nuclear receptors whose physiological ligands have not yet been identified. Although ERRs are closely related to estrogen receptors (ERs) they do not respond to estrogens . ERRs share a comparable modular structure with the classical ligand-regulated nuclear receptors such as steroid hormone receptors, thyroid hormone receptors, retinoid receptors, and vitamin D receptor. They thus consist of a centrally located conserved DNA-binding domain (DBD), a carboxyl-terminal ligand-binding domain (LBD), and a variable amino-terminal domain (NTD). The LBD folds into a three-layered sandwich structure composed of 12 α-helices (H1–12) forming a hydrophobic ligand-binding pocket . In liganded nuclear receptors, ligand binding induces a conformational change in the LBD switching it from an inactive to an active conformation that allows interactions with transcriptional coactivator proteins. Interactions between the LBD and coactivator proteins involve the H12 containing the activation function-2 (AF-2) . In PNU-120596 molecular to the liganded nuclear receptors, structural studies have confirmed that the LBDs of ERRγ and ERRα adopt an active conformation and interact with coactivators in the absence of any bound ligand , . In line with this, ERRs activate transcription constitutively in reporter gene assays . Their transcriptional activities are enhanced by the coactivator PGC-1 that was proposed to act as a protein ligand for ERRs . In addition, the activity of ERRγ has also been shown to be modulated by cross-talk with other transcription factors , , . Taken together, these observations suggest that the activities of ERRs are regulated by coactivator concentration or cross-talk with other transcription factors rather than by ligand binding. The recently published crystal structures demonstrated that the ligand-binding pockets of ERRα and ERRγ are extremely small compared to the pockets in liganded nuclear receptors because the ERR ligand-binding pockets are partly filled by lipophilic side chains , . Ligand binding was predicted to displace H12 from the transcriptionally active conformation and thus to have an inhibitory rather than an activating effect on ERRs. Indeed, most of the identified synthetic ERR ligands act as inverse agonists. The ER agonist diethylstilbestrol (DES) deactivates all three ERRs and the selective ER modulator 4-hydroxytamoxifen (4-OHT) binds to and inactivates ERRβ and ERRγ , , . The crystal structures of the ERRγ LBD in complex with DES and 4-OHT showed that binding of these inverse agonists resulted in rotation of the side chain of F435 and that the new rotamer of F435 displaced H12 from its agonistic position , . Recently, certain flavonoids were reported to inhibit the transcriptional activity of ERRγ by suppressing the interaction between ERRγ and the coactivator PGC-1α or by promoting the degradation of PGC-1α . Zuercher et al.  reported however the identification of a synthetic ERRγ agonist GSK4716. Binding of GSK4716 to ERRγ caused rearrangements in the LBD that allowed the formation of a larger ligand-binding pocket and subsequently, ligand binding did not displace H12. Binding of GSK4716 led to global stabilization of the ERRγ LBD . This demonstrated that it is possible to design and identify agonists for ERRγ. Although most ERRα ligands act as inverse agonists, certain phytoestrogens have been shown to be ERRα agonists . Because of similarities between the ERR-family members, we have investigated whether these plant estrogens were also able to stimulate the transcriptional activity of ERRγ. Phytoestrogens are plant-derived polyphenolic non-steroidal compounds that have estrogenic properties . The main classes of phytoestrogens are lignans and isoflavones. Lignans, found in cereals, fruits, and vegetables, are metabolized by intestinal bacteria to enterolactone and enterodiol. Soybeans are a rich source of isoflavones. The principal isoflavones are genistein and daidzein. Daidzein is further converted to equol by gut microflora but this conversion has a high inter-individual variability . Equol has been shown to have higher estrogenic activity than the precursor compound daidzein . The interest in phytoestrogens has been increasing due to their suggested beneficial health effects. Phytoestrogen rich diet appears to reduce the risk of cardiovascular disease, osteoporosis as well as breast and prostate cancer .