Structural Basis for the Deactivation of the Estrogen-related Receptor γ by Diethylstilbestrol or 4-Hydroxytamoxifen and Determinants of Selectivity

Abstract

The estrogen-related receptor (ERR) ␥ behaves as a constitutive activator of transcription. Although no natural ligand is known, ERR␥ is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERR␥ ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERR␥ results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ER␣ LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERR␥ does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERR␥ and ER␣ reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERR␥ relative to ER␣. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERR␥ and reveal structural differences with ERs that explain why ERR␥ does not bind estradiol or raloxifene and will help to design new selective antagonists.The estrogen-related receptors ERR␣, 1 ERR␤, and ERR␥ (NR3B1, -2, and -3) (1) form a subfamily of orphan nuclear receptors that share significant amino acid homology with the estrogen receptors ER␣ and ER␤ (NR3A1 and -2) (2, 3). Because of the high conservation in the DNA binding domain, ERRs and ERs have overlapping DNA binding selectivity (4 -6) and, accordingly, may co-regulate target genes in tissues in which they are co-expressed. ERR subfamily members have for example been shown to modulate the expression of ER target genes in bone (7,8) or breast tissue (9, 10). Importantly, overexpression of ERR␣ and ERR␥ in samples from breast cancer patients correlates with unfavorable and favorable biomarkers, respectively (11). Therefore, these receptors might serve as prognostic markers themselves or even be targets for endocrine therapy in human breast cancer.Despite their significant homology with ERs in the ligand binding domain (LBD), ERRs do not (or only very weakly) respond to estradiol (E2) (2, 12). Furthermore, whereas ERs are ligand-activated receptors, ERRs are constitutively active (13-16), and a structural study confirmed that the ERR␥ LBD can adopt a transcriptionally active conformation and interact with the steroid receptor coactivator 1 (SRC-1) in the absence of any ligand (12). Together, these observations suggest that ERRs are ligand-independent activators of transcription whose activation potential may rather be determined by the presence of transcriptional coactivators (17)(18)(1...

X-ray Crystal Structures of the Estrogen-related Receptor-γ Ligand Binding Domain in Three Functional States Reveal the Molecular Basis of Small Molecule Regulation

“…1) diethylstilbestrol (1) and 4-hydroxytamoxifen (2, 4-OHT) profile as ERR␥ inverse agonists (10). X-ray crystal structures of 1 and 2 bound to ERR␥ have been reported, and both structures are consistent with the observed inverse agonism (11). Due to the small volume of the ligand pocket, the receptor undergoes a large conformational change upon binding 1 or 2 that displaces the AF-2 helix resulting in a loss of coactivator binding.…”

Nuclear receptors, mitochondria and lipid metabolism

“…Because these receptors have very small ligand biding pockets, endogenous ligand discovery remains unlikely, although synthetic ligands have been developed and ERRα is blocked by diethylstilbestrol, while ERRβ and γ are blocked by tamoxifen (Coward et al 2001;Greschik et al 2002;Willy et al 2004). The activity of ERRs appears to be constitutive, with the ligand binding pocket stably arranged in an active conformation without ligand (Xie et al 1999;Greschik et al 2002;Greschik et al 2004). Because ERRα is widely expressed in adult tissues, especially in tissues that utilize or can utilize fatty acid β-oxidation, many studies have addressed its role in cellular energetics (Luo et al 2003).…”

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value

“…Therefore, the transcriptional activity of ERR does not depend on the presence of an endogenous agonist. The crystal structure of the ERR -DES complex showed that the binding of DES to ERR results in a conformational change of a single phenylalanine residue (Phe435) which partially fills the ligand-binding pocket in the apo-LBD and thus interferes with the position of His12 in the transcriptionally active LBD conformation (Greschik et al, 2004;Fig. 5d).…”