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

Abstract

X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-␥ (ERR␥) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERR␥ LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERR␥ seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERR␥ LBD with 4-hydroxytamoxifen (4-OHT), and the ERR␥ LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERR␥ plus 4-OHT complex. The observed agonist-bound state contains the ERR␥ LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERR␥ by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix. The nuclear receptor (NR)2 superfamily comprises 48 ligand-regulated transcription factors in the human genome.Amino acid sequence alignment identifies several conserved regions within the NRs: a central, highly conserved DNA binding domain that allows for the sequence-specific recognition of DNA in the promoter region of the genes; a C-terminal ligand binding domain (LBD) that is able to bind small, lipophilic molecules and influence the interaction with cofactors involved in transcriptional regulation; and a poorly conserved N-terminal domain. X-ray crystallography has revealed that the canonical NR LBD is composed of 10 -13 ␣-helices arranged in a three-layered sandwich, the interior of which varies in volume and functional character to define the molecular basis of ligand specificity.The estrogen-related receptors (ERR␣, ERR␤, and ERR␥, or NR3B1, NR3B2, and NR3B3, respectively) define a subfamily of three orphan NRs most closely related to the classic estrogen receptors (ER␣ and ER␤). Because of high sequence conservation within the DNA binding domains of the two subfamilies, the classic ERs and orphan ERRs are able to recognize common DNA binding sites (also known as response elements) proximal to target genes (1). For example, the orphan ERRs have demonstrated control over classic ER target genes in breast (2) and bone (3). Althou...

Involvement of estrogen-related receptors in transcriptional response to hypoxia and growth of solid tumors

“…The anticancer efficacy of the existing ERR inhibitor (i.e., DES) holds great promise for developing new ligands with increased potency and selectivity. The crystal structures of ERR LBDs have revealed how inhibitors can promote dissociation of coactivators and potential recruitment of corepressors (17,33). This detailed knowledge of the structural mechanism sets the stage for the rational design and rapid identification of new compounds with desired properties for cancer therapy.…”

Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ

“…2c,d), consistent with ligand effects on other nuclear receptor LBDs, for which it typically increases the T m by 1-6°C (refs. 30,31 ). These results suggested that a change in protein conformation occurs on heme binding to REV-ERB LBDs, and this idea was supported by peptide interaction 'conformation sensing' assays ( Supplementary Fig.…”

Intrinsic Protein Disorder and Protein-Protein Interactions

“…The antagonist further reinforces the binding of corepressor to the nuclear receptor. MoRF mechanisms are also involved in the down regulation of target gene expression when the nuclear receptor corepressor 2 binds to related nuclear receptors such as peroxisome proliferator activated receptor (PPAR), estrogen related receptor gamma (ERR-gamma) and progesterone receptor (PR) [20][21][22].…”