Structural Pharmacology of Estrogen-Related Receptors

Abstract

Estrogen-related receptors (ERRs) are orphan nuclear receptors critical to the regulation of energy metabolism, mitochondrial biogenesis, and tissue-specific transcriptional programs. This review provides a comprehensive structural analysis of ERR isoforms (ERRα, ERRβ, and ERRγ), emphasizing insights from X-ray crystallography and NMR studies. We discuss the ligand-binding domains (LBDs), coactivator and corepressor interactions, and the molecular mechanisms underlying ligand-induced agonism or antagonism. Structural comparisons with estrogen receptors (ERs) reveal key amino acid determinants for ligand selectivity and functional activity. Furthermore, we highlight the development of isoform-selective synthetic ligands, including inverse agonists such as GSK5182, DN200434, and DN201000, with therapeutic potential in metabolic, neurodegenerative, and oncologic diseases. This synthesis of structural data provides a framework for rational drug design targeting ERRs, supporting the development of selective modulators to manipulate ERR signaling in a tissue- and disease-specific manner.

Graphical Abstract

INTRODUCTION

Estrogen Related Receptors (ERRs) represent the first identified orphan nuclear receptors within the NR3B subgroup of the nuclear receptor superfamily. Initially discovered in 1988 during a search for steroid hormone receptors homologous to estrogen receptors (ERα and ERβ), ERRα was named for its structural similarity to ERs despite lacking affinity for estrogens.¹ The ERR subfamily comprises three isoforms, ERRα (NR3B1), ERRβ (NR3B2), and ERRγ (NR3B3), which share ~60% sequence homology with ERs, particularly in the DNA-binding domain (DBD) and ligand-binding domain (LBD).² Structurally, ERRs adopt the canonical nuclear receptor architecture, featuring 12 α-helices in the LBD that form a hydrophobic ligand-binding pocket (LBP).³ Unlike ERs, ERRs lack endogenous ligands and exhibit constitutive transcriptional activity independent of estrogen binding,^4,5 relying on interactions with coactivators like peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) to regulate gene expression.⁶ This ligand-independent activation underscores their unique role as metabolic sensors in energy-demanding tissues.

ERRs are ubiquitously expressed but are enriched in tissues with high oxidative capacity, including skeletal muscle, heart, brain, liver, and adipose tissues.^1,7,8 In these tissues, ERRs regulate the expression of genes critical for mitochondrial biogenesis, oxidative phosphorylation, fatty acid oxidation, and the tricarboxylic acid (TCA) cycle,^9–13 thereby maintaining cellular energy homeostasis.^14,15 For instance, in skeletal muscle, ERRα is indispensable for mitochondrial function and repair. Murine models with skeletal muscle-specific ERRα deletion exhibit impaired mitochondrial biogenesis, disrupted AMP-activated protein kinase (AMPK) signaling, and delayed regeneration following injury.^9,13,16–18 Conversely, transgenic overexpression of ERRγ in murine skeletal muscle enhances mitochondrial content, elevates oxidative enzyme activities (e.g., citrate synthase), and augments exercise endurance, even in the absence of physical training.^19–23 This suggests that ERRγ can drive a shift toward a more oxidative muscle fiber phenotype, improving metabolic efficiency and endurance capacity.

Consistent with its shared tissue distribution pattern with ERRγ, ERRβ is highly expressed in skeletal muscle and found in other metabolically active tissues (e.g., retina, spinal cord, heart, muscle, and kidney), albeit often at lower levels than ERRγ.^24,25 While its physiological roles remain less defined, ERRβ is implicated in neural development, synaptic plasticity, and neuroprotection, with knockout models displaying embryonic lethality due to placental defects.²⁶ This isoform-specific expression highlights the functional diversity of ERRs across tissues.

The central role of ERRs in energy metabolism positions them as compelling therapeutic targets for metabolic, neurodegenerative, and neoplastic diseases. Modulation of ERR activity could offer novel approaches to treating conditions such as obesity, diabetes, and muscle degenerative diseases by enhancing mitochondrial function and energy expenditure. In metabolic disorders, ERRα agonism might have beneficial effects on mitochondrial function through the suppression of hepatic gluconeogenesis, offering potential benefits for obesity and type 2 diabetes.¹³ In neurodegeneration, ERRα activation reduces AD progression by upregulating amyloid-beta clearance pathways, while ERRγ modulates tau phosphorylation, suggesting neuroprotective roles.²⁷ Conversely, ERRα inhibition is under investigation in breast and prostate cancers, where its overexpression drives tumorigenesis via metabolic reprogramming of glycolysis and glutaminolysis.^28–31 Emerging roles for ERRs in age-related macular degeneration (AMD) further underscore their therapeutic relevance, with ERRα influencing pathogenic pathways in retinal cells.³² Additionally, ERRγ’s capability to induce oxidative muscle remodeling suggests its potential for enhancing endurance capacity and treating muscle-wasting disorders.

ERRs function as metabolic transcriptional hubs that integrate environmental, developmental, and physiological cues to regulate energy metabolism. Their isoform-specific expression patterns, divergent physiological roles, and tissue-selective functions underscore the importance of developing selective modulators capable of targeting individual ERRs without eliciting off-target effects. From a pharmacological perspective, a deeper understanding of the conformational dynamics of the ERR LBD and the influences of coactivator/ligand binding is essential to the rational design of agonists, antagonists, and inverse agonists of ERRs. Despite being classified as orphan receptors, several synthetic ligands and modulators have now been identified, some of which show isoform selectivity and hold promise for therapeutic development.

The diverse biological actions of the ERR subfamily extend far beyond metabolic regulation, underscoring their involvement in a wide spectrum of clinical disorders. ERRα, the most extensively characterized isoform, governs oxidative metabolism and mitochondrial function in energy-demanding tissues but also contributes to pathophysiological processes underlying metabolic syndrome, type 2 diabetes, and nonalcoholic fatty liver disease, largely through dysregulation of the PGC-1α/ERRα axis. Overexpression of ERRα has been associated with tumor metabolic reprogramming and poor prognosis in breast, ovarian, and prostate cancers, emphasizing its oncogenic role in promoting glycolytic and glutaminolytic fluxes that sustain tumor growth.^12,33

In contrast, ERRβ (ESRRB) exhibits more restricted expression but plays pivotal roles in embryonic development, stem-cell pluripotency, and mitotic regulation. Its splice variants (e.g., ERRβ2) have been implicated in cell-cycle control and tumor differentiation, with altered expression observed in breast, prostate, and bladder cancers.^34,35 Emerging evidence indicates that ERRβ plays an important role in neural development and photoreceptor neuroprotection, with studies showing its involvement in maintaining neuronal integrity and survival, particularly within the visual system and developing central nervous tissues.³⁶

ERRγ (ESRRG), while sharing structural similarity with ERRα, functions as a master regulator of mitochondrial biogenesis, oxidative metabolism, and iron homeostasis in cardiac, hepatic, and neural tissues. Dysregulated ERRγ signaling contributes to insulin resistance, hepatic steatosis, and cardiac hypertrophy, while recent studies implicate it in neurodegenerative diseases such as Parkinson’s disease, where ERRγ modulates mitochondrial and synaptic gene networks that influence vulnerability to synucleinopathy.³⁷ Additionally, ERRγ activity has been linked to vascular calcification and atherosclerosis through BMP2-dependent signaling pathways. ³⁸

Taken together, these findings establish ERRs as critical regulators that bridge mitochondrial metabolism, cell proliferation, and disease pathogenesis across multiple organ systems. Their isoform-specific actions, metabolic and oncogenic (ERRα), developmental and mitotic (ERRβ), and neuro-cardiometabolic (ERRγ), underscore the therapeutic potential of selectively modulating ERR signaling in diverse clinical contexts, ranging from metabolic and cardiovascular disorders to cancer and neurodegeneration.

The aim of this review is to provide a comprehensive summary of all X-ray crystallographic and NMR structures of ERR isoforms reported in the literature, elucidating key features of their DBDs, LBDs, and interaction interfaces with coactivators or corepressors. By summarizing current knowledge on ERR structure and function, we hope to enhance the understanding of these transcriptional regulators and to support ongoing efforts in drug discovery targeting ERRs for the treatment of metabolic, neurodegenerative, oncologic, and muscular diseases.

STRUCTURE OF ERRS

The structural architecture of ERRs, characterized by canonical nuclear receptor domains, underpins their constitutive activity and isoform-specific functions. Sequences highlighting key structural motifs and residue variations across ERRα, ERRβ, ERRγ, and ERα are shown in Figure 1a. This review comprehensively surveys experimentally determined structures of ERR isoforms, with all X-ray crystallographic structures and NMR structures of ERR complexes discussed herein listed in chronological order in Table 1. The corresponding UniProt accession numbers and source organisms of these proteins are summarized in Table 2. Unless otherwise noted, all ERR sequences discussed in this review correspond to Homo sapiens. One study employed Mus musculus ERRγ, which is identical in sequence to the human ERRγ protein, thereby justifying crossspecies comparison. These structural data reveal critical insights into LBP topology, mechanisms of ligand-independent activation, coactivator recruitment (notably PGC-1α), and the conformational changes induced by agonists, inverse agonists, and environmental ligands. Additionally, chemical structures of all ligands discussed in this review are shown in Figure 1b. Key findings include the role of conserved phenylalanine clusters in filling the ERRα LBP, the divergent helix 12 (H12) displacement mechanisms governing inverse agonism across isoforms, and the structural basis for ligand selectivity between ERRs and ERα.

Figure 1.

(a) Sequence alignment of the LBD sequences of human ERα, ERRα, ERRβ, and ERRγ. Amino acid residues that are conserved across the receptors are highlighted in yellow. Residue numbering corresponds to each individual sequence. The degree of conservation at each aligned position across the four receptors is shown by the color bar above the alignment: warm colors (e.g., red) indicate high conservation, while cool colors (e.g., blue) represent low conservation. (b) Chemical structures of ERR ligands discussed in this review.

Table 1.

Available ERR X-ray Structures

Receptor	Peptide	Ligand	PDB ID	Resolution	Year	Ref.
ERRγLBD	SRC-1	-	1KV6	2.70 Å	2002	39
ERRβDBD	-	DNA	1LO1	-	2002	40
ERRγLBD	SRC-1	-	1TFC	2.40 Å	2004	41
ERRγLBD	-	DES	1S9P	2.13 Å	2004	41
ERRγLBD	-	4-OHT	1S9Q	2.20 Å	2004	41
ERRγLBD	-	4-OHT	1VJB	3.20 Å	2004	41
ERRαLBD	PGC-1α	-	1XB7	2.50 Å	2004	42
ERRγLBD	-	GSK5182	2EWP	2.30 Å	2005	43
ERRγLBD	-	-	2GP7	2.45 Å	2006	44
ERRγLBD	RIP140	-	2GPO	1.95 Å	2006	44
ERRγLBD	RIP140	GSK4716	2GPP	2.60 Å	2006	44
ERRγLBD	-	4-OHT	2GPU	1.70 Å	2006	44
ERRγLBD	SMRT	4-OHT	2GPV	2.85 Å	2006	44
ERRγLBD	-	BPA	2E2R	1.60 Å	2007	45
ERRα(C325S)LBD	-	CPD1a	2PJL	2.30 Å	2007	46
ERRγLBD	-	ClCH3Ph	2P7A	2.30 Å	2007	47
ERRγLBD	-	BPA	2P7G	2.10 Å	2007	47
ERRγLBD		4-OHT	2P7Z	2.50 Å	2007	47
ERRγLBD	-	4-α-cumylphenol	2ZAS	2.00 Å	2007	48
ERRγLBD	-	-	2ZBS	1.80 Å	2007	48
ERRαLBD	PGC-1α Box3	-	3D24	2.11 Å	2008	49
ERRαLBD	-	Diaryl ether-based thiazolidinedione (TZD) 29	3K6P	2.00 Å	2011	50
ERRγLBD	-	DN200434	5YSO	2.50 Å	2017	51
ERRγLBD	-	BPB	6I61	1.65 Å	2018	52
ERRγLBD	-	HPTE	6I62	1.65 Å	2018	52
ERRγLBD	-	BPA	6I63	2.23 Å	2018	52
ERRγLBD	-	BPE	6I64	1.91 Å	2018	52
ERRγLBD	-	4-iso-propylphenol	6I65	1.50 Å	2018	52
ERRγLBD	-	4-sec-butylphenol	6I66	1.60 Å	2018	52
ERRγLBD	-	tetrahydro-2-naphthol	6I67	1.75 Å	2018	52
ERRγLBD	-	DN201000	6A6K	2.90 Å	2018	53
ERRγLBD	-	DN200699	6KNR	2.80 Å	2019	54
ERRγLBD	-	BPA-monoF	6K3N	1.97 Å	2019	55
ERRβ(Y215H)LBD	SRC2–2	BPA	6LIT	2.00 Å	2020	56
ERRβ(Y215H)LBD	PGC-1α-2a		6LN4	2.61 Å	2020	56
ERRαLBD	PGC-1α	DS45500853	7E2E	2.70 Å	2021	57
ERRγDBD	-	DNA, Pla2g12b promoter	8IFO	2.20 Å	2023	58
ERRγDBD	-	6-nitroindole (6NI)	9KNC	1.90 Å	2025	59
ERRγDBD	-	indole	9KND	1.52 Å	2025	59
ERRγDBD	-	5-nitroindole	9KNE	1.80 Å	2025	59
ERRγDBD	-	4028691	9KNF	1.62 Å	2025	59
ERRγDBD	-	4034496	9KNG	1.50 Å	2025	59

Table 2.

Uniprot Accession Numbers and Source Organisms of the Proteins Discussed in This Review

Protein	Organism	Uniprot ID
ERRα	Homo sapiens	P11474
ERRβ	Homo sapiens	O95718
ERRγ	Homo sapiens	P62508
ERRγ41	Mus musculus	P62509

ERRα: STRUCTURAL AND FUNCTIONAL INSIGHTS

Structural Features of ERRα LBD.

In 2004, Kallen et al. reported the first X-ray crystal structure of LBD of ERRα complexed with a peptide derived from peroxisome proliferator-activated receptor coactivator-1α (PGC-1α).⁴² The cavity volume of ERRα LBP is 100 ų, less than half the volume reported for ERRγ LBP (220 Å^{3 39}; see Figure 2a). This significant decrease is primarily attributed to the presence of Phe328 and Val491 in ERRα, corresponding to Ala272 and Ala431 in ERRγ, respectively (Figure 2b). Notably, Phe328 is essential for the constitutive activity of ERRα.⁶⁰ Furthermore, the substitution of Tyr326 and Asn346 in ERRγ with Phe382 and Gly402 in ERRα, respectively, creates additional space within ERRα LBP, allowing residues Phe382, Ala393, Gly402, and Leu405 to move closer together. LBP of ERRα is practically filled with an “internal ligand”, predominantly comprising a cluster of phenylalanine side chains (Phe328, Phe382, Phe495, and Phe510; highlighted in Figure 2c). This feature underlies ERRα’s ligand-independent transcriptional activity. The nearly filled LBP not only prevents ERRα from binding to E2 completely but also causes its binding to diethylstilbestrol (DES) energetically unfavorable, resulting in a weak binding affinity for DES (Figure 2c).

Figure 2.

(a) X-ray structure of ERRα LBP complexed with PGC-1α (PDB ID: 1XB7). (b) Comparison of the LBD of ERRα (gold) and ERRγ (pink; PDB ID: 1S9P). The hypothetical position of DES is determined by the superposition with the complex ERα-DES LBD (PDB ID: 3ERD). (c) Representation of ERRα LBD complexed with DES hypothetically positioned in its LBP. The phenylalanine cluster (highlighted in red) forms an “internal ligand” that almost filled ERRα LBD. The steric hindrance between Phe495 and the hypothetical DES helps explain its weak binding affinity to ERRα. (d) Close-up view of PGC-1α (steel) bound to the coactivator binding site of ERRα LBD. The hydrogen bonds formed by the “charge clamp” Lys340 and Glu512 are shown as black lines. (e) Superimpositions of apo ERRα LBD (gold; PDB ID: 1XB7) and ERRα-1A complex (pink; PDB ID: 2PJL). Compound 1A is shown in gray. (f) Comparison of ERRα with PGC-1α coactivator (left) and ERRα-1A complex (right). Color changes from blue at the N-terminal to red at the C-terminal. PGC-1α peptide is shown in gray, and compound 1A is in steel. (g) Superimpositions of ERRα-1A (blue) and ERRα-29 (gold; PDB ID: 3K6P) complexes. Compound 29 is shown in purple, while compound 1A is omitted for clarity. The thioether bond is represented by a dotted line.

The coactivator peptide PGC-1α exhibits a unique structure featuring an inverted LXXLL motif (i.e., a LLXYL motif). It makes the canonical “charge clamp” interactions with Lys340 of H3 and Glu512 of H12 in ERRα. PGC-1α, containing this nuclear box motif form the L3 site (Figure 2d), demonstrates strong binding to ERR LBD with a k_d of 0.6 μM; in contrast, the binding affinity of a peptide containing SRC-1 motif to ERRα LBD is markedly weaker (k_d = 88 μM), as determined by a fluorescence polarization-based binding assay. The diminished binding affinity of SRC-1 can be attributed to amino acid differences when compared to PGC-1α, resulting in less favorable interactions and steric hindrance. Additionally, differential scanning calorimetry scans reveal that all three leucine-rich regions of PGC-1α contribute to its binding to ERRα LBD.

Inverse Agonism in ERRα: Structural Mechanism.

Kallen et al.⁴⁶ reported the first X-ray structure of ERRα LBD (with C325S mutation) bound with an inverse agonist, cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (referred to as compound 1A hereafter). To address biochemical instability issues during protein purification and crystallization linked to cysteine oxidation, the authors introduced a C325S mutation in ERRα-LBD. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) measurements confirmed that the C325S mutation did not alter ERRα conformation significantly, with comparable thermodynamic binding parameters between wild-type ERRα-LBD and the C325S mutant for compound 1A (k_d values of 770 and 930 nM, respectively). Furthermore, NMR spectroscopy analysis of compound 1B binding to wild-type ERRα-LBD in solution revealed no discrepancies in observed relaxation effects compared to the crystal structure of the compound 1A complex.

The binding of compound 1A induced a sequence of conformational changes in the LBP, unveiling a novel mechanism of H12 displacement. Notably, movement of the N-terminal of H3 prompted residues Val321 and Leu324 to shift from their original positions, thus creating additional space for the ligand accommodation (Figure 2e). Furthermore, a reorientation of Phe328 (H3) led to the displacement of Phe510 (H12), causing H12 to bind at the activation function-2 (AF-2) site and directly competing with the binding of the coactivator PGC-1α (Figure 2f). The altered rotamer of Phe328 allows Glu331 to reorient toward the ligand, forming a salt bridge and a water-mediated hydrogen bond with compound 1A.

The conformational change of ERRα to inverse agonist binding is quite different compared to ERRγ. For ERRγ, antagonism induced by ligands 4-hydroxytamoxifen (4-OHT) and DES arises from the reorientation of Phe435 (H11).^41,43,44 The rotamer of Phe435 sterically interferes with H12 in its agonist position, resulting in complete dissociation of H12 (Figure 5b and e). Conversely, for ERRα, the transition of H12 from its agonist position to the coactivator groove is derived by the reorientation of Phe328. This repositioning of H12 into the coactivator groove is also observed for ERα complexes with selective ER modulators (SERMs) such as raloxifene or 4-OHT (Figure 5d).^61,62

Figure 5.

(a) Crystal structure of the homodimer ERRγ LBD (PDB ID: 1KV6). The ERRγ LBD adopts a transcriptionally active conformation after binding to SRC-1 coactivator peptide (depicted in red). Superimposition of X-ray structures of (b) ERRγ apoLBD (PDB ID: 1TFC) and ERRγ-DES (PDB ID: 1S9P), (c) ERRγ-DES and ERRγ-4-OHT (PDB ID: 1S9Q), (d) ERRγ-DES and ERα-DES (PDB ID: 3ERD), and (e) ERRγ-4-OHT and ERα-4-OHT (PDB ID: 3ERT). ERRγ apoLBD is shown in gold, ERRγ-DES in pink, ERRγ-4-OHT in green, and ligand-bound ERα complexes in blue. Only portions of the structures are shown for clarity. (f) Overlay of X-ray structures of ERRγ-GSK5182 (khaki; PDB ID: 2EWP) and ERα-4-OHT (blue; PDB ID:3ERT) complexes. Hydrogen bonding between GSK5182 and the Y-N pair of ERRγ is absent in ERα, which has two phenylalanine residues instead. Ligand 4-OHT is omitted for clarity. (g) X-ray structure of ERRγ-RIP140 (PDB ID: 2GPO) with two distinct pockets. Superimpositions of unliganded (h) ERRγ-RIP140 (khaki) and ERRγ-GSK4716-RIP140 (pink; PDB ID: 2GPP) complexes, and (i) liganded ERRγ complexed with RIP140 coactivator (pink) and ERα-4-OHT complex (blue; PDB ID:3ERT).

Binding and stabilization of H12 at the AF-2 site of ERα has been attributed to its characteristic LXXLL motif.^61,62 Interestingly, in ERRα complex, H12 assumes a distinct, well-defined position within the coactivator groove despite the absence of this canonical LXXLL “coactivator” motif.

Patch et al.⁵⁰ identified a series of diaryl ether-based thiazolidinediones as potent inverse agonists of ERRα using a high-throughput Thermofluor⁶³ binding screen, followed by systematic Structural-Activity Relationship (SAR) optimization. Among these ligands, compound 29 (Figure 1b) emerged as the lead candidate, exhibiting strong inhibitory activity in both FRET and two-hybrid luciferase reporter assays, with IC₅₀ of 0.04 ± 0.015 μM and 0.6 ± 0.21 μM, respectively. Compound 29 displayed high microsomal stability, minimal cytochrome P450 (CYP) inhibition, and marked selectivity (>50-fold) for ERRα over ERRγ. It also showed weak binding to ERα (IC₅₀ = 2.2 μM) and ERβ (IC₅₀ = 0.6 μM) without disrupting the estradiol-mediated coactivator recruitment or inducing estrogenic activity in cellular or in vivo assays. Importantly, compound 29 exhibited negligible activity against other nuclear receptors, including PPARs, LXRs, and RARα, differentiating it from traditional thiazolidinedione (TZD) insulin sensitizers.

The X-ray crystallographic structure of ERRα LBD complexed with compound 29 revealed substantial conformational rearrangements consistent with inverse agonism. Helix 12 was displaced into the coactivator groove formed by helices H3, H4, and H12, similar to that observed in the ERRα-1A complex.⁴⁶ Compound 29 interposes between Phe328 and Phe495, disrupting their π-π stacking interaction by displacing Phe495, while its vanilloid ring forms π-contact with Phe328 and van der Waal interactions with Phe495 (Figure 2g). The 2-trifluoromethyl substituent occupies a hydrophobic pocket delineated by Met362, Val366, Leu398, Leu401, Leu405, Val491, Phe495, and Met506, accounting for its enhanced potency relative to methyl-substituted analogues. The TZD headgroup of compound 29, on the other hand, projects toward the solvent-exposed surface and interacts with residues from a symmetry-related molecule in the crystal lattice. Notably, a thioether bond is formed between Cys325 (H3) and the α,β-unsaturated TZD moiety of compound 29 via conjugate addition.

LC-MS analysis further showed that the ERRα-29 complex forms covalently but dissociates in a slow yet reversible manner, with an estimated half-life of approximately 18 h. Pharmacokinetic studies demonstrated high oral bioavailability (>78%) and broad systemic exposure (AUC_0-n ≈ 30–220 μg·h/mL) across rat, mouse, dog, and monkey species. In diet-induced obesity mice and diabetic rats, oral administration of compound 29 normalized serum triglyceride and insulin levels, improved glucose tolerance, and restored insulin sensitivity without affecting body weight. Gene expression analysis indicated enhanced fatty-acid oxidation and mitochondrial function, mimicking the metabolic phenotype of ERRα knockout mice. These findings highlight the therapeutic potential of selective ERRα modulation for the treatment of metabolic disorders such as type 2 diabetes.

PGC-1α Coactivator Binding to ERRα.

Greschik et al. determined the X-ray crystal structure of ERRα LBD in complex with a PGC-1α box3 peptide (204RRPCSELL-KYLTTNDD219) and provided evidence for the communication between the ERRα homodimer interface and the PGC-1α binding surface via the helix 8–9 loop.⁴⁹ The structure revealed interactions involving amino acids Arg205, Pro206, and Cys207 of PGC-1α with H4, the H8–H9 loop, and H12 of ERRα, which were not included in a previously reported structure (Figure 3a and b).⁴² Specifically, Arg205 forms a hydrogen bond with Gln262 (H4), and water-mediated hydrogen bonds with Ser337 (H8-H9 loop) and Ala420 (H12). Additionally, Asp423 at the C-terminal of the LBD interacts with His341 (H8-H9) and Arg205 of PGC-1α.

Figure 3.

X-ray crystal structures of ERRα LBD complexed with (a) PGC-1α box3 peptide (PDB ID: 3D24) and (b) mutant PGC-1α box3 peptide (PDB ID: 1XB7). (c) X-ray crystal structure of ERα LBD complexed with TIF-2 box3 peptide (PDB ID: 1GWR). ERRα are shown in a) gold and b) blue, coactivator PGC-1α in gray, ERα in navy, and TIF-2 in yellow. (d) Superimposition of apo-ERRα (PDB ID: 1XB7) and ERRα-DS45500853. Residues Leu324, Cys325, and Phe328 move away from the LBP to accommodate binding of DS45500853. (e) X-ray crystal structure of ERRα-DS45500853 (PDB ID: 7E2E). The ligand forms extensive hydrophobic interactions with the lipophilic LBP, as well as water-mediated hydrogen bonding interactions with residues Arg372 and the backbone of Phe382. Apo-ERRα is shown in pink, ERRα-DS45500853 in gold, and DS45500853 in blue.

To perform functional analysis, two-hybrid interaction studies were applied in transiently transfected eukaryotic cell lines. In this two-hybrid system, wild-type and mutant nuclear receptor-interacting regions of PGC-1α, including the entire interaction domain (ID), box2, and box3, were expressed in fusion with the Gal4 DNA-binding domain, while the ERRα LBD was fused with the VP16 activation domain. Through this assay, it was observed that PGC-1α efficiently interacts with ERRα via a canonical LXXLL (box2) and an atypical LLKYL (box3) motif. A single point mutation in either box2 at ID (L144A) or box3 at ID (L210A) reduces the interaction between ERRα LBD and PGC-1α, while a double point mutation of L144A and L210A eliminates it completely, highlighting their essential role in binding. Examining the PGC-1α box3 at four residues (202PQRR205), two double point mutations (P202A & Q203A and R204A & R205A) revealed that while the PQ mutations do not affect the interactions, the RR to alanine mutations reduce the binding to minimal levels. This indicates the crucial role of N-terminal amino acids at position −6 to −3 (Arg204 and Arg205) in binding of PGC-1α box3 to ERRα LBD.

The authors investigated which regions of the ERRα surface are responsible for the specific binding of PGC-1α. Mutating two residues in the coactivator (CoA) cleft, M258V and Q262E in ERRα, significantly decreased the interaction between the mutant ERRα and PGC-1α ID, box2, and box3 (Figure 3a and b). The substantial loss of box2 binding was surprising, given that the ERα LBD is known to interact efficiently with the canonical LXXLL region of PGC-1α (Figure 3c).⁶⁴ Therefore, ERRα (M258V/Q262E) was expected to acquire properties similar to ERα. Consequently, the authors proposed that Met258 and Gln262 contribute to the overall binding affinity but do not determine coactivator binding selectivity.

However, swapping the H8-H9 loop of ERRα with that of ERα completely eliminates the interaction with PGC-1α, the reverse substitution enabling ERα to interact significantly with PGC-1α box3. Additionally, mutation of Asp338 (H8-H9 loop) or Arg315 to alanine in the ERRα homodimer interface significantly affects PGC-1α binding, as well as in vitro homodimeric DNA binding and coactivator interaction. This implies that the H8-H9 loop of ERRα allosterically connects the homodimer interface with the coactivator cleft, explaining the preferential binding of PGC-1α to ERRα homodimer over its monomer.

Finally, the study examined whether mutations in the H8–H9 loop region influence DNA or CoA binding of full-length ERRα by evaluating complex formation in the presence of labeled double-stranded oligonucleotides with two different motifs (two direct AGGTCA repeats without spacing (DR0) or two inverted AGGTCA repeats with three-base pair spacing (ERE)) through Electrophoretic Mobility Shift Assays (EMSAs). The results showed homodimeric binding of ERRα to both DR0 and ERE motifs. However, mutation in the H8-H9 loop (R315A or D338A) of ERRα led to monomer binding to the DR0 motif. When tested with PGC-1α, although PGC-1α could upshift DNA-bound ERRα, all DNA-bound mutants were less efficiently shifted by PGC-1α ID. Thus, mutations in the H8–H9 loop such as R315A or D338A influence not only homodimerization but also CoA binding of DNA-bound, full-length ERRα.

Discovery of ERRα Agonists.

Shinozuka et al.⁵⁷ identified novel ERRα agonists through SAR studies of high-throughput screening (HIT) hits, leading to the discovery of benzimidazole derivative DS20362725 (compound 3d) and acetophenone analogue DS45500853 (compound 5c) (Figure 1b). Both compounds function as micromolar ERRα agonists while avoiding peroxisome proliferator-activated receptor (PPARγ) transcriptional activity, a critical advancement given the undesired PPARγ activation associated with TZD-based derivatives. In the ERRα luciferase reporter assay, DS20362725 exhibited an EC₅₀ of 1.1 μM, whereas DS45500853 displayed an EC₅₀ of 5.4 μM.

X-ray crystallography of ERRα LBD in complex with DS45500853 and the PGC-1α coactivator peptide revealed structural mechanisms of agonism. The ERRα LBD adopts an active conformation upon DS45500853 binding, with significant rearrangements in helix H3: residues Leu324, Cys325, and Phe328 shift by up to 5.4 Å to accommodate the ligand (Figure 3d). Rearrangement of Phe328 was also observed in molecular dynamics (MD) simulations of ERRα bound to an agonist compound.^65,66 DS45500853 engages the hydrophobic LBP via van der Waals interactions and forms a water-mediated hydrogen bond between its phenolic hydroxyl group and residues Arg372 and Phe382 (Figure 3e). This interaction underscores the necessity of the hydroxyl group for binding potency, as its removal abolished activity. DS20362725, designed by eliminating the TZD moiety from initial hits, retained ERRα agonism (EC₅₀ = 1.1 μM) while avoiding PPARγ activation, though its structural details were not resolved.

ERRβ: LIGAND AND DNA BINDING MECHANISMS

Structural Characterization of ERRβ Ligand-Binding Domain.

Yao et al.⁵⁶ achieved a breakthrough in studying the ERRβ by engineering a single-residue mutation (Y215H) in its LBD, which drastically improved solubility and stability when expressed in E. coli while preserving transcriptional activity comparable to the WT receptor. This advancement enabled the first structural and biochemical characterization of ERRβ, a previously elusive orphan nuclear receptor. Functional assays revealed that ERRβ recruits the steroid receptor coactivator-2 (SRC2–3) motif in response to the agonist endocrine disruptor 2,2-bis(4-hydroxyphenyl)propane (BPA) with an EC₅₀ ~ 300 nM and is antagonized by 4-OHT with an IC₅₀ ~ 280 nM (Figure 1b). These affinities are weaker than those observed for ERRγ, which exhibits higher sensitivity to BPA (EC₅₀ = 160 nM) and 4-OHT (IC₅₀ = 50 nM), while ERRα shows no detectable activity toward either ligand.

The X-ray crystallographic structure of apo-ERRβ LBD reveals a constitutively active conformation of the AF-2 helix, stabilized by hydrophobic interactions involving residues such as Leu240, Leu243, Leu251, and Phe425. These interactions mimic ligand-induced stabilization, explaining ERRβ’s ligand-independent transcriptional activity (Figure 4a). In the BPA-bound ERRβ structure, ligand binding induces conformational rearrangements: residue Glu250 forms a hydrogen bond with BPA’s hydroxyl group, while Phe410 shifts spatially to accommodate the ligand (Figure 4b). BPA also forms a hydrogen bonding with residue Tyr301 in ERRβ (Tyr326 in ERRγ). Notably, ERRγ’s higher affinity for BPA is attributed to an additional hydrogen bond between BPA and Asn346, which is replaced by Tyr321 in ERRβ, whose hydroxyl group orients away from BPA to minimize steric interference (Figure 4c). Similarly, 4-OHT antagonism in ERRγ is reinforced by hydrogen bonding between Tyr326 and Asn346, optimizing the LBP environment (Figure 4d). In contrast, ERRα’s inability to bind either ligand is due to steric hindrance caused by the bulky Phe328 side chain, highlighting key structural determinants of subtype specificity.

Figure 4.

(a) X-ray crystallographic structures of ERRβ-LBD complexed with the coactivator PGC-1α (PDB ID: 6LN4). The LBD is stabilized by the hydrophobic sidechains from pocket residues in the absence of ligands. (b) Superimposition of X-ray structures of ERRβ-LBD and ERRβ-BPA (PDB ID: 6LIT). Ligand-binding induces conformational changes of residues Glu250 and Phe410. Apo-ERRβ is shown in blue, ERRβ-BPA in navy blue, PGC-1α in red, and BPA in yellow. Superimposition of X-ray crystallographic structures of apo-ERRα (golden; PDB ID: 1XB7), ERRβ-BPA (blue; PDB ID: 6LIT): (c) apo-ERRγ (khaki; PDB ID: 1KV6), (d) 4-OHT-ERRγ (khaki; PDB ID: 2GPU). Bindings of both ligands are hindered by the residue Phe328 in ERRα. Ligand BPA is shown in yellow and 4-OHT in pink. Hydrogen bonds are represented using dashed lines. (e) Superimposition of X-ray crystallographic structures of apo-ERRα (golden; PDB ID: 1XB7), ERRβ-BPA (blue; PDB ID: 6LIT), and apo-ERRγ (khaki; PDB ID: 1KV6). The coactivator PGC-1α is shown in green. Hydrophobic interactions between residue Tyr213 and hydrophobic residues from the ERRs are shown via lines. NMR structure of hERRβ-DNA complex of (f) superposition of 20 ensembles and (g) a single ensemble (PDB ID: 1LO1). The strands of DNA are shown in gray while the bases are in blue. The core DBD of hERRβ that makes major-groove interactions with the DNA is shown in yellow, and the CTE, which is responsible for minor-groove interactions, is in pink. Residues 187 – 194 are excluded for clarity.

ERRβ, like its subtypes ERRα and ERRγ, exhibits a conserved ability to interact with both canonical LXXLL motifs (e.g., SRC1–2, SRC2–3, and SRC3–3) and the atypical LLKYL motif of PGC-1α-2. The X-ray structure also sheds light on ERRβ’s coactivator selectivity, which arises from conserved hydrophobic interactions between residue Tyr213 in PGC-1α-2 and residues Val252, Ile255, and Leu424 in ERRβ. The conservation of these hydrophobic residues across all three ERRs explains their shared high binding affinity for PGC-1α-2 (Figure 4e).

Monomeric DNA Recognition by ERRβ: Role of the C-Terminal Extension.

Gearhart et al. reported NMR solution structure of the human ERRβ-DNA complex.⁴⁰ Differing from typical nuclear receptors which bind to DNA as dimers, the hERRs bind to DNA as monomers.⁶⁷ The core of DBD, defined by the zinc finger fold spanning residues 97–168, employs a mechanism similar to that of other nuclear receptors by engaging the major groove of DNA (Figure 4f and g). Additional hydrogen bonding and electrostatic interactions between the core DBD and the phosphate backbone within the major groove further enhance the binding. The C-terminal extension (CTE), defined by residues 169–194, plays a crucial role in contributing to high binding affinity. It features an AT-hook-like motif capable of engaging with both AT base pairs and sequences containing GC. While the T-box residues align with the DNA’s phosphate backbone, the A-box delves deep into the DNA’s minor groove, forming an extensive hydrophobic core.

The stability of CTE and the ERRβ-DNA complex structures are reinforced by the CTE’s interaction with both the minor groove of the DNA and the core DBD, traditionally involved in dimerization. To explore the stabilizing role of CTE in the complex, truncations and single-site mutations were investigated, and the binding affinity of ERRβ to DNA was assessed using gel shift assays. Truncation at Lys186 demonstrated minimal effect on DNA binding. On the other hand, the Y185A mutation resulted in a significant reduction in DNA binding affinity, while truncation at Arg174 led to a complete loss of binding capability, highlighting the pivotal role of ERRβ’s CTE in ERRβ-DNA binding.

ERRγ: STRUCTURE, LIGAND INTERACTION, AND DNA RECOGNITION

ERRγ Structural Characteristics and Coactivator Interactions.

In 2002, Greschik et al. reported the first X-ray crystal structure of the LBD of the ERRγ complexed with SRC-1 peptide.³⁹ The crystal structure of human ERRγ (hERRγ) was derived using the homodimer of the human estrogen receptor alpha (hERα) LBD/Glucocorticoid Receptor Interacting Protein 1 (GRIP1) Nuclear Receptor (NR) box complex as the initial model.⁶² It reveals that ERRγ LBD shares a similar dimerization mode as that of hERα LBD, featuring a contact area of 1245 ų per molecule. The LBD of ERRγ consists of 12 α helices (H1-H12) and exhibits a standard three-layered α-helical sandwich structure (Figure 5a). Its putative LBP is bounded by a sequence of 22 amino acids, enclosing a small cavity volume of 200 ų. Remarkably, the structure of ERRγ LBD bound with SRC-1 peptide assumes a transcriptionally active conformation even in the absence of any ligands.

Molecular docking of the previously reported ERR antagonists, DES and 4-OHT, are impeded from binding ERRγ LBD in its active conformation due to steric clashes with Leu345 in H7 or Phe435 in H11 of ERRγ. This indicates that structural rearrangement into an antagonist LBD conformation is necessary upon binding to DES and 4-OHT. The nearly identical ligand binding cavities of ERRγ and ERRβ, differing only by two amino acids (Val313 vs. Ile288 and Asn346 vs Tyr321 between ERRγ and ERRβ, respectively), indicate that both can bind similar ligands (Figure 1a). Larger differences of amino acids are observed between ERRα and ERRβ/ERRγ. Notably, the presence of phenylalanine in ERRα, replacing alanine in both ERRβ and ERRγ, significantly alters the size and shape of the ERRα pocket.

Greschik and colleagues further substantiated that the ERRγ interaction with SRC-1 is independent from the binding of ligands. They utilized native polyacrylamide gel electrophoresis (PAGE) to track the interaction between ERRγ-LBD and the receptor interaction domain of its coactivator SRC-1, as well as the conformational changes induced by antagonists. In the absence of antagonists, ERRγ-LBD effectively bound to SRC-1 to become a complex. However, in the presence of antagonists such as DES or 4-OHT, the migration of ERRγ-LBD on native gel is retarded, indicating a significant conformational change of ERRγ-LBD.

Mutations were introduced to the ERRγ-LBD protein to either obstruct or enlarge its ligand binding cavity. The results indicated that these mutations did not affect the interaction between ERRγ-LBD and SRC-1 in the absence of antagonists. However, cavity-blocking mutations nullified the effects of antagonists, enabling interaction between the mutant ERRγ-LBD and SRC-1. Interestingly, cavity-enlarging mutations only helped ERRγ-LBD overcome the antagonist effect induced by DES, not by 4-OHT. The authors speculated that the disparity in the conformational changes induced by DES and 4-OHT could be attributed to the long side chain of 4-OHT (Figure 1b).

Additional experiments were conducted in variant cell lines to further support the notion that ERRγ can activate transcription independently of an endogenous ligand. In these experiments, wild-type (WT) and mutant ERRγ-LBD were fused to the DNA binding domain (DBD) of Gal4, and Gal4-ERRγ-LBD fusion proteins were evaluated for their transcriptional activation potential in transient transfection assays using synthetic reporters in eukaryotic cell lines. The results from these experiments were consistent with those obtained from the native PAGE studies.

Antagonist-Induced Conformational Changes in ERRγ.

Greschik et al. reported the crystal structure of ERRγ LBD complexed with antagonist ligands, DES and 4-OHT.⁴¹ The result provided molecular insight into the conformational changes occurring upon binding of antagonists to ERRγ and explained why ERRγ, unlike ERs, does not bind estradiol (E2) or raloxifene (RAL).

Upon ligand binding, such as DES or 4-OHT, there occurs a rotation of the side chain of Phe435, thereby inhibiting the formation of the canonical transcriptionally active conformation of ERRγ LBD and compromising coactivator recruitment (Figure 5b and c). In ERα-DES complexes, minimal conformational adaptation is required upon binding to DES due to the replacement of Phe435 with Leu525. This structural alteration does not interfere with the agonist position of H12 (Figure 5d). The distinct structural features of the LBP between ERRγ and ERα complexed with 4-OHT (Figure 5e) are primarily responsible for the insensitivity of ERRγ to E2 or the selective estrogen receptor modulator (SERM) RAL. The deeper protrusion of E2 and RAL in the LBP, compared to 4-OHT, cannot be accommodated in an energetically favorable manner due to the spatial constraints imposed by the presence of Leu345 in ERRγ (Ile424 in ERα) and a shifted position of H7 relative to ERα.

Development of ERRγ Inverse Agonists.

Drawing upon insights provided by X-ray structures of 4-OHT bound ERRγ⁴¹ and ERα,⁶² Chao et al. utilized 4-hydroxytamoxifen (4-OHT) as a scaffold to craft and synthesize inverse agonists with enhanced selectivity for ERRγ.⁴³ Notably, the residue differences, namely Tyr326 and Asn346 in ERRγ, (which correspond to Phe404 and Phe425 in ERα), suggest the possibility of hydrophilic interactions between the former receptor and the ethyl chain of 4-OHT, revealing crucial determinants for enhancing ERRγ selectivity. Building upon 4-OHT as a scaffold, among the array of compounds synthesized, compound 15, known as GSK5182 (Figure 1b), exhibited a remarkable 25-fold increase in binding selectivity for ERRγ over ERα. Furthermore, the X-ray structure, published by the same research group, verified the existence of hydrogen bonding interactions between GSK5182 and the amino acid residues Tyr326 and Asn346 (Figure 5f). These interactions are absent in ERα due to the substitution of the Y-N pair present in ERRγ with nonpolar phenylalanine groups (Phe404 and Phe425, respectively). This key structural difference highlights a potential pathway for enhancing ERRγ selectivity.

Binding affinity was assessed through a scintillation proximity binding assay (radioligand displacement) using [3H] labeled 4-OHT or estradiol, revealing that GSK5182 displayed a 25-fold enhancement in binding selectivity for ERRγ compared to ERα. Furthermore, a coregulator peptide of receptor interacting protein 140 (RIP140) bounded ERRγ complex Homogeneous Time-Resolved Fluorescence (HTRF) assay was employed to gauge alterations in receptor-coactivator interaction due to the ligands. Results indicated that GSK5182 decreased this interaction with lower potency than 4-OHT. In addition, cell-based luciferase assays were conducted using HeLa cell lines to evaluate the effects of the 4-OHT analogs. At a concentration of 1 μM, GSK5182 suppressed ERRγ transcriptional activity by 69%, while exerting no significant impact on ERα.

Ligand Binding Modes and Pocket Flexibility in ERRγ.

Wang et al.⁴⁴ investigated the X-ray crystal structures of ERRγ LBD in three states, revealing five distinct configurations: unliganded (with and without a coregulator peptide), inverse agonist bound (with and without a peptide), and agonist bound with a coregulator. No significant differences were observed between the apo receptor and ERRγ-RIP140 complex. Notably, the crystal structure of apo ERRγ LBD uncovered an additional pocket alongside the previously identified 280 ų LBP (220 ų as reported in ref.39 ). This newly discovered pocket, separated from the first LBP defined by Glu275 and Arg316, exhibited a volume of 390 ų (Figure 5g).

The structure of inverse agonist, 4-OHT, bound ERRγ LBD confirms the rearrangement of Phe435 and displacement of H12 as observed by Greschik et al.⁴¹ This rearrangement effectively prevents the binding of coactivator peptides. Little structural change of ERRγ LBD was observed upon additional peptide binding of the silencing medicator of retinoid and thyroid hormone action protein (SMRT) to ERRγ•4-OHT complex. The organization of molecules within the asymmetric unit of ERRγ•4-OHT•SMRT complex is quite interesting. This unit comprises six molecules, three homodimers, wherein only one molecule in each dimer exhibits a completely disordered H12, while the other one maintains a well-ordered structure. The interaction between the well-ordered H12 and the disordered H12 in a neighboring homodimer forms a tetramer, of which the molecules are related by a noncrystallo-graphic 2-fold axis.

The overall crystal structure of the agonist GSK4716 bound ERRγ-RIP140 closely resembles that of unliganded ERRγ. No rotation of Phe435 or Phe450 from their apo conformation was observed. Instead, rearrangement of H1-H3 of the receptor, induced by the displacements of Pro246, Glu247, and Lys248, was detected. Additionally, the reorientation of the phenol-binding residues Glu275 and Arg316 leads to the disruption of the salt bridge formed between them and facilitates access to the second LBP (Figure 5h). The rearrangement of helices and reorientation of residues collectively forms a single, enlarged LBP with a volume of 610 ų, which is sufficiently large to accommodate GSK4716 without necessitating displacement of H12.

Two crucial structural factors are identified as significantly influencing the binding of GSK4716 to ERRγ: the polar interaction with Asp328 and the small, nonpolar hydrazone fragment.^44,68 Structural analogs of GSK4716 featuring nonpolar phenol replacements exhibit drastic reduction in potency. Conversely, introduction of large or more polar hydrazone fragments eliminates the binding completely.

GSK4716 demonstrates binding to ERRγ within the submicromolar range while showing no affinity for ERα or ERβ at concentrations up to 50 μM. One contributing factor is the hydrogen bond formation between the phenolic ring of GSK4716 and Asp328 in ERRγ, located near the receptor surface. Such interaction is absent in ERα due to the presence of Pro406 at the corresponding position (Figure 5i). In ERRγ, the phenol binding residue Glu275 undergoes a rotation and interacts with Glu247, whereas in ERα, the corresponding residue Pro325 fails to stabilize the glutamic acid rotamer. In addition, GSK4716 interacts with another phenol binding residue Arg316 in ERRγ through a water molecule. The less energetically favorable rotations of phenol binding residues in combination with the absence of a phenol binding site for the alternative binding mode may explain the binding selectivity of GSK4716 for ERRγ over ERα.

To elucidate the mechanism of increased transcriptional activity of ERRγ upon agonist ligand binding, a thermal sensitivity assay was conducted using circular dichroism (CD). The results showed that the ligand GSK4716 induced a smaller increase in protein stability (t_1/2 increased by 0.9 ± 0.1 °C) compared to the coactivator RIP140 LXXLL peptide (t_1/2 increased by 3.0 ± 0.2 °C). However, their effects on protein stability were additive when both the peptide and agonist ligand were added to the ERRγ LBD (t_1/2 increased by 4.2 ± 0.2 °C). In contrast, the adverse ligand 4-OHT exhibited a higher increase in protein stability (t_1/2 increased by 6.7 ± 0.1 °C) without any additive effect compared to the agonist ligand. This is mainly because, in the ERRγ-4-OHT structure, the AF-2 helix is positioned in a way that prevents all coregulator binding.⁴⁴

BPA Binding and Functional Effects in ERRγ.

Endocrine disruptor Bisphenol A or BPA (Figure 1b)^69–73 has been identified as binding to ERRγ with high affinity (k_d = 5.5nM) while maintaining ERRγ’s basal constitutive activity.⁴⁵ Matsushima et al. elucidated the molecular mechanism by determining the X-ray crystal structure of ERRγ-BPA complex. The X-ray diffraction analysis revealed that ERRγ-LBD crystallized in a homodimeric form, and that BPA binding does not interfere with homodimer formation. This homodimer formation of purified ERRγ-LBD was also demonstrated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, showing two peaks: the monomer peak emerged around 27,600 while the dimer peak was found around 55,200. Superimposing the apo ERRγ LBP and ERRγ-BPA complex revealed minimal structural changes in the receptor upon binding (Figure 6a). Consequently, H12 maintains its active conformation, which explains the preservation of ERRγ’s constitutive activity.

Figure 6.

(a) Superimposition of apo ERRγ (khaki; PDB ID: 1TFC) and ERRγ-BPA complex (pink; PDB ID: 2E2R). H12 in the complex is highlighted in red, and BPA is in gray. (b) Close-up view at ERRγ-BPA LBP. (c) Superimposition of BPA bound (pink; PDB ID: 2P7G) and ClCH3Ph bound (sky blue; PDB ID: 2P7A) ERRγ. Ligand BPA is shown in gray and ClCH3Ph in yellow. Hydrogen bonds are indicated by black lines. (d) Superimposition of ERRγ-BPA (pink) and ERRγ-SRC (yellow; PDB ID: 1KV6). Minimal conformational changes of the ERRγ main chain and its coactivator binding site are observed when binding to BPA, which explains the functional silence of the ligands. (e) Superimposition of ERRγ LBD structures in complex with 6NI (PDB ID: 9KNC), indole (PDB ID: 9KND), 5-nitroindole (PDB ID: 9KNE), 4028691 (PDB ID: 9KNF), and 4034496 (PDB ID: 9KNG), each cocrystallized with the corepressor peptide RIP40 (navy blue). Helix H12 remains in its active conformation across all structures. Ligands are omitted for clarity. (f) superimposition of ligand-binding poses of ERRγ in complex with indole analogs. The hydrogen-bonding network among ligand, receptor residues, and the water molecule (W1) are represented as lines. (g) X-ray crystallographic structure of ERRγ-LBD complexed with 4-α-cumylphenol (PDB ID: 2ZAS). Dotted lines indicate interactions between the ligand and the protein receptor. (h) Superimposition of ERRγ-LBD complexed with 4-α-cumylphenol (golden) and BPA (pink; PDB ID: 2E2R). (i) Superimposition of X-ray crystallographic structures of BPA (PDB ID: 2E2R) and BPA-monoF (PDB ID: 6K3N) bound ERRγ. Additional ligandprotein interactions of ERRγ-(BPA-monoF), compared to ERRγ-BPA, are indicated using black dotted lines. ERRγ is shown in khaki, BPA in gray, and BPA-monoF in purple.

The stabilization of BPA binding in the ERRγ LBP is facilitated by the key amino acid Tyr326 (β-strand 1) through two robust interactions: a T-shaped π/π interaction with benzene ring A, where one of the edges or vertexes of the benzene ring points toward the π face of the corresponding aromatic ring, and an OH/π interaction with benzene ring B of BPA (Figure 6b). In addition, CH/π interaction is observed between ring A of BPA and the isobutyl-methyl group of Leu309 (Figure 6b), resulting in a sandwiched hydrophobic interactions of ring A with Leu309 and Tyr326. The stability of BPA within the LBP is further enhanced by the formation of hydrogen bonding between BPA’s B ring and Asn346, in conjunction with the OH/π interaction with Tyr326. Moreover, binding of BPA is reinforced by the CH/π interactions and molecular interactions between the two methyl groups of BPA and Phe435 and Met306, respectively.

In the ERRγ-BPA complex, the position and conformation of H12 closely resemble those of the apo form of ERRγ LBD. Notably, H12 within the complex maintains its activation conformation in the absence of SRC-1 coactivator. Furthermore, BPA demonstrates inverse antagonist properties against the inverse agonist 4-OHT, effectively reversing the deactivation caused by 4-OHT in ERRγ. This suggests that BPA has the potential to reposition H12 from its inactive conformation to its active state.

A saturation binding assay was conducted with WT ERRγ-LBD and its mutants, where Glu275, Met306, Leu309, Arg316, Tyr326, Asn324, and Phe435 were replaced with alanine, were assessed using saturation binding assay with tritium-labeled BPA. Most of the mutant receptors exhibited reduced specific binding and weakened binding affinity. The assay showed that the simultaneous substitution of amino acid residues Glu275 and Arg316 resulted in a complete loss of specific binding, indicating that these two residues are critically important for BPA binding to the pocket.

Functional Silence of ERRγ Ligands.

Abad et al. screened approximately 3000 chemical compounds against the LBD of ERRγ using ThermoFluor.⁴⁷ ThermoFluor assay, a miniaturized thermal shift assay that monitors the fraction of unfolded protein as a function of temperature, it was shown that both BPA and ClCH₃Ph interact with ERRγ and enhance its stability. Similar to the compound 4-OHT, the presence of BPA and ClCH₃Ph increased the stability of ERRγ by 4.0 to 5.7 °C. The authors further conducted time-resolved fluorescence resonance energy transfer (TR-FRET) experiments to examine the functional response of ERR ligands. It was found that neither BPA nor ClCH₃Ph displaced the coactivator peptide bound to the receptor, unlike 4-OHT, which displaced the peptide fragment with an affinity of approximately 11 nM. In the presence of 4-OHT, both BPA and ClCH₃Ph competed with 4-OHT and allowed the formation of the ERRγ-SRC2 complex with EC₅₀ values of 120 and 500 nM, respectively. Binding of BPA and ClCH₃Ph was also observed through isothermal titration calorimetry, characterized by highly exothermic binding enthalpies (ΔH ~ −21 kcal/mol) and affinities of 70 and 380 nM, respectively.

However, the authors could not demonstrate an enhancement in the receptor’s affinity for coactivator peptide fragments or a change in transcriptional activity in trans-activation cell-based assays. Therefore, they concluded that both ligands are potent for ERRγ and competitive for binding at the 4-OHT binding site but are functionally silent.

The receptor ERRγ was cocrystalized with BPA and ClCH₃Ph (Figure 1b) to investigate the molecular basis of the functionally silent nature of these ligands. Crystal structures of the resulting complexes revealed that the hydroxyl groups of both ligands form hydrogen bonds with residues Glu275 and Arg316 (Figure 6c). Interestingly, in ERRγ-BPA complex, a three-way hydrogen bonding interaction was observed among residues Tyr326, Asn346, and the hydroxyl group of the second BPA phenyl ring. Note residue Asn346 is not conserved among NRs, making the N346-OH interaction specific to ERRγ. The additional ligand–receptor interactions in ERRγ-BPA compared to ERRγ-ClCH₃Ph account for the significantly higher binding affinity of BPA toward ERRγ relative to ClCH₃Ph.

Superimposition of ERRγ-SRC and ERRγ-BPA structures (Figure 6d) showed minimal conformational changes in the main chain and conserved structures at the coactivator binding site, providing structural evidence for the functional silence of the two ligands. Additionally, superimposing the crystal structures of ERRγ-BPA and ERRγ-4-OHT supports the finding by Greschik et al.⁴¹ that the reorientation of Phe435, which forces H12 out of its agonist position and becoming disordered, is crucial for ERR’s inhibition.

A recent study by Shuai et al. identified indole and its methylate derivative skatole, two metabolites derived from the gut microbiota, as potential endogenous ligands of ERRγ.⁵⁹ Biochemical assays showed that both compounds directly bind to the ERRγ LBP with high affinity (k_d ≈ 1–2 μM), stabilizing the protein (ΔT_m ≈ + 4 °C). Structural-activity analysis of indole analogs revealed that substitutions at the 3-position abolished binding, whereas 5- or 6-nitro substitutions enhanced affinity (Figure 1b). Among these, 6-nitroindole (6NI) exhibited the strongest binding (k_d ≈ 0.8 μM), highlighting the critical role of the indole ring and its hydrogen-bonding orientation within the compact hydrophobic pocket.

Functionally, indole and skatole act as competitive ligands within ERRg’s LBP, modulating receptor activity in the presence of known agonists and antagonists. Despite their minimal intrinsic transcriptional activity, both effectively neutralize the agonistic effects of GSK4716 by occupying the same binding site. Interestingly, indole attenuates the antagonistic efficacy of 4-OHT and GSK5182, partially restoring transcriptional activity, whereas skatole, displaying weak inverse agonism, suppresses the agonist activity of the endocrine disruptor BPA.

X-ray crystallographic structures of ERRγ LBD bound to indole and several analogs, cocrystallized with a corepressor peptide RIP140, revealed that all ligands occupy the hydrophobic LBP without displacing the C-terminal activation helix H12, which remains in an active conformation (Figure 6e). Their binding is coordinated by a conserved hydrogen-bonding network mediated by a structural water molecule (W1) bridging Glu275 and Arg316, while the indole nitrogen forms an additional hydrogen bond with the backbone carbonyl of Leu268, anchoring the ligand within the pocket (Figure 6f).

Although these static structures show minimal rearrangement of H12, MD simulations elucidated the dynamic mechanism for differential ligand activity. The high-affinity analog 6NI forms a persistent hydrogen bond between its nitro substituent and the W1 network, transmitting subtle strain that increases the flexibility of H12 and destabilizes the coactivator-binding surface, which is consistent with 6NI’s strong antagonistic activity. In contrast, indole, lacking a stabilizing substituent, exhibits positional fluctuations within the pocket and induces negligible perturbation of H12, resulting in its neutral functional profile. Together, these findings delineate a dynamic pharmacophore in which 6-position modifications enhance affinity and promote antagonism through allosteric destabilization of H12, whereas bulkier substitutions at the 2- or 3-position abolish binding.

In cell-based luciferase assays, both indole and skatole exhibited little direct transcriptional regulation. However, RNA-seq analysis of ERRγ-expressing cells treated with indole revealed differential expression of 11 genes among 133 ERRγ targets, including CYP1A1, PLIN4, ABCA1, and IFIT family members. These results suggest potential cross-talk between ERRγ and other indole-responsive signaling pathways, such as the aryl hydrocarbon receptor (AhR) network.

Structural-Activity Relationships of BPA Analogues.

Bisphenol A has been shown to bind to ERRγ while retaining the receptor’s constitutive basal activity.^74,75 Matsushima et al. discovered that 4-α-cumylphenol (Figure 1b), which contains only one of the phenol-hydroxyl groups present in BPA, exhibits comparable potency to BPA in reversing the deactivation of ERRγ activity by 4-OHT. In contrast, the absence of phenol-hydroxyl group in diphenylproprane (Figure 1b) completely eliminates its activity in the receptor-binding affinity assays, underscoring the critical role of the phenolic moiety in receptor interaction.⁴⁸

To quantify binding ability, 4-α-cumylphenol was tested in a luciferase reporter gene assay and compared with BPA, 4-OHT, and 2,2-diphenylpropane. Among these, BPA, 4-α-cumylphenol, and 4-OHT exhibited nearly identical potency in binding to ERRγ, with IC₅₀ values of approximately 10 nM. In contrast, 2,2-diphenylpropane was essentially inactive (IC₅₀ > 10,000 nM).

To investigate the significance of the phenol-hydroxyl group and to elucidate its role in ligand binding of ERRγ, Matsushima et al. determined the X-ray crystal structures of ERRγ complexed with BPA and with 4-α-cumylphenol.^45,48 The phenol-hydroxyl group of 4-α-cumylphenol binds to ERRγ LBD similarly to BPA, forming hydrogen bonds with residues Glu275 and Arg316. In addition, it interacts hydrophobically with Met306, Leu309, Tyr326, Leu345, and Phe435 (Figure 6g). Notably, the cumylphenyl group of 4-α-cumylphenol engages in C-H/π interactions with Tyr326 and Leu345, while BPA’s B-ring hydroxyl group hydrogen-bonds with Asn346.

X-ray structures of the ERRγ complexes highlighted the induced-fit behavior and conformational flexibility of Leu345 as a key finding (Figure 6h). In the BPA-bound structure, the isobutyl side chain of Leu345 reorients to accommodate the B-ring hydroxyl group. Conversely, when complexed with 4-α-cumylphenol, this same side chain forms hydrophobic interactions with the ligand with minimal overall conformational changes relative to its apo structure.

Suyama et al.⁵⁵ systematically evaluated the effects of halogenation on BPA binding to ERRγ. Halogenated BPA analogs generally exhibited reduced binding affinity and transcriptional activity compared to BPA, with fluorinated analogs (e.g., BPA-monoF, BPA-diF) being notable exceptions. Competitive binding assays revealed IC₅₀ values of 5.30 nM for BPA-monoF and 5.95 nM for BPA-diF, comparable to BPA (6.45 nM). In contrast, bulkier halogen substituents (Cl, Br, I) or increased halogenation (tri/tetra-substitution) drastically reduced ERRγ binding, with IC₅₀ values exceeding 1 μM for most analogs (e.g., BPA-triCl: 473 nM, BPA-tetraF: 111 nM).

X-ray crystallography of the BPA-monoF/ERRγ-LBD complex demonstrated a binding mode nearly identical to BPA, with hydroxyl groups forming hydrogen bonds to Glu275, Arg316, and Asn346. Critically, the fluorine atom of BPA-monoF engaged in a noncovalent interaction with Asn346, enhancing binding affinity (Figure 6i). Fragment molecular orbital (FMO) calculations quantified this interaction, revealing a total binding energy of −79.4979 kcal/mol for BPA-monoF, driven by strong interfragment interactions with Glu275 (−23.7199 kcal/mol), Arg316 (−7.6594 kcal/mol), and Asn346 (−12.7554 kcal/mol).

Reporter gene assays in HeLa cells showed that halogenated analogs lacked inverse agonistic activity but retained inverse antagonistic effects in the presence of 4-OHT. BPA-monoF and BPA-diF exhibited EC₅₀ values of 1.58 ± 0.21 μM and 4.13 ± 0.32 μM, respectively, restoring ERRγ transcriptional activity displaced by 4-OHT. Tri- and tetra-halogenated analogs showed negligible activity, underscoring the steric constraints of ERRγ’s LBP.

In addition, Suyama et al. observed a strong correlation between the EC₅₀ values from competitive binding assays and the binding energies obtained through docking calculations for halogenated BPA analogs (cf. Figure 8 of ref. 55 ). These findings suggest that the binding affinity of BPA analogs can be predicted using computational methods such as docking calculations.

Thouennon et al.⁵² systematically characterized the interaction of environmental endocrine-disrupting chemicals (EDCs) with ERRγ, identifying 4-sec-butylphenol (EC₅₀ = 109 nM) and bisphonel-E (BPE; EC₅₀ = 142 nM) as the most potent agonists among the bisphenol, alkylphenol, and naphthol derivatives tested (Figure 1b). BPE exhibited the highest binding affinity (k_d = 48.7 nM), followed by BPA (k_d = 98.6 nM), corroborating earlier reports of BPA’s agonistic activity.^47,48,74,75

Structural analyses of ERRγ’s LBD in complex with these compounds revealed conserved binding motifs. Bisphenols engaged both phenol groups: one formed hydrogen bonds with Arg316 and Glu275, while the other interacted with Asn346, stabilized by a Tyr326-Asn346 hydrogen bond (Figure 7a-c). In contrast, alkylphenols and naphthols relied on a C-H/π interaction with Phe435 alongside polar contacts with Arg316 and Glu275. Strikingly, ligand binding preserved ERRγ’s transcriptionally active conformation, with helix H12 remaining in its agonist position, indistinguishable from the apo structure (Figure 7d).⁴⁸ Molecular dynamics simulations further demonstrated that BPA and BPE rigidified ERRγ’s LBD, notably reducing conformational fluctuation in loop L9-L10 (cf. Figure 4c of ref.52 ), suggesting ligand-induced stabilization rather than structural reorganization.

Figure 7.

Interactions of ERRγ LBD and compounds from the (a) bisphenol (BPE; PDB ID: 6I64), (b) alkylphenol (4-sec-butylphenoll; PDB ID: 6I66), and (c) naphthol (tetra-2-hydronaphthol, THN; PDB ID: 6I67) families. Hydrogen bonds are represented by black dashed lines. Ligands BPE, 4-s-butylphenol, and THN are shown in orange, blue, and turquoise, respectively. (d) Superimposition of apo-ERRγ (dark khaki; PDB ID: 2ZBS) and ERRγ-BPA (light blue; PDB ID: 6I63). Ligand BPA is shown as sphere. (e) Superimposition of ERRγ complexed with DN200434 (pink; PDB ID: 5YSO) and GSK5182 (blue; PDB ID: 2EWP). Additional π-H interaction between DN200434 and Phe435 is shown via dotted line. (f) X-ray crystallographic structure of ERRγ-DN201000 (PDB ID: 6A6K). Ligand-protein interactions are indicated using black dotted lines. ERRγ is shown in khaki and DN201000 in pink. (g) X-ray crystallographic structure of ERRγ-compound 25 (PDB ID: 6KNR). Ligand-protein interactions are indicated using black dotted lines. ERRγ is shown in khaki and compound 25 in orange. (h) X-ray crystallographic structure of ERRγ DBD in complex with G12Bpro element (PDB ID: 8IFO). ERRγ DBD bound to the downstream and upstream are shown in blue and yellow, respectively. Zinc ions are depicted as spheres. (i) Superimposition of ERRγ DBD-G12Bpro (yellow) and ERRβ DBD-ERRE (purple) complexes; the ERRE is omitted for clarity. (j) Close-up view of the dimer interface in the ERRγ DBD-G12Bpro complex. The downstream and upstream monomers are shown in blue and yellow, respectively. Hydrogen bonds are indicated by dotted lines.

Functional validation using a stable HG5LN cell line expressing GAL4-ERRγ(LBD) and a luciferase reporter demonstrated that the inverse agonist 4-OHT (IC₅₀ = 458 nM) suppressed basal ERRγ activity by 50%, while BPA (EC₅₀ = 174 nM) enhanced it by 125%. Co-treatment experiments revealed BPA’s ability to competitively reverse OHT-mediated repression, underscoring ligand binding as a key regulatory mechanism.

Thermal shift and ITC assays corroborated the cellular data, with ligand binding increasing ERRγ-LBD stability (ΔT_m up to + 6.3 °C for 4-sec-butylphenol) and exhibiting favorable enthalpy terms (e.g., ΔH = −21.2 kcal/mol for BPA). These results highlight a unique activation mechanism where environmental ligands stabilize ERRγ’s preexisting active conformation through selective rigidification of dynamic regions, rather than inducing classical H12 repositioning.

DN200434 and DN201000 as ERRγ Inverse Agonists.

Singh et al.⁵¹ demonstrated elevated ERRγ expression in poorly differentiated anaplastic thyroid cancer (ATC) compared to normal thyroid tissues via tissue microarrays (TMAs) and immunohistochemistry (IHC). The inverse correlation with sodium iodide symporter (NIS) expression underscores ERRγ’s potential as a therapeutic target for restoring radioiodine avidity in refractory ATC.

They identified a novel orally bioavailable ERRγ inverse agonist DN200434 through optimization of a 300-compound library derived from the hit compound GSK5182⁴³ (Figure 1b). DN200434 exhibited exceptional selectivity of ERRγ (binding IC₅₀ = 0.04 μM) over ERRα, ERRβ, and ERα with a functional potency (IC₅₀ = 0.006 μM) 12-fold greater than GSK5182. The X-ray structure of ERRγ-DN200434 complex revealed a binding mode similar to that of ERRγ-GSK5182 (Figure 7e), which was stabilized by hydrogen bonds with residues Tyr326, Asn346, Glu275, alongside electrostatic interaction with Asp273. DN200434’s enhanced potency relative to GSK5182 is attributed to the additional π-H interaction with Phe435. Luciferase reporter assays of ERRγ mutants (D273A, E275A, Y326A, and N346A) confirmed the critical role of Asp273 in mediating ligand–receptor interactions, as alanine substitution abolished inhibitory effects for both DN200434 and GSK5182.

In vitro studies in CAL62 ATC cells demonstrated DN200434’s dual functionality: dose-dependent apoptosis induction (evidenced by cleaved PARP/caspase-3 upregulation and Annexin V/PI staining) and restoration of NIS-mediated radioiodine uptake. At 12 μM, DN200434 increased iodide avidity 2.7-fold within 24 h, a response abolished by the NIS inhibitor KClO₄. Mechanistically, DN200434 upregulated NIS, TSHR, TPO, and TG mRNA/protein levels while activating MAP kinase signaling (p-ERK1/2), which was suppressed by MEK inhibitors (PD98059 and U0126). Notably, DN200434 also redifferentiated ATC cells, downregulating glucose transporters (GLUT-1/GLUT-4) and reducing F-18-FDG uptake by 1.7-fold, suggesting metabolic reprogramming toward a differentiated phenotype.

In vivo, oral DN200434 (200 mg/kg) enhanced radioiodine incorporation in ATC xenografts 4.4-fold, as quantified by ¹²⁴I-PET/CT imaging. Immunoblotting and IHC confirmed NIS membrane localization in treated tumors. Combination therapy with DN200434 and ¹³¹I significantly suppressed tumor growth in mouse models without adverse effects, highlighting its translational potential for radioiodine-refractory ATC.

Kim et al.⁵³ developed DN201000 (compound 35, Figure 1b), a tetrasubstituted olefin analogue and potent inverse agonist of ERRγ, through systematic SAR and structure–property relationships (SPR) optimization. Building on the scaffold of the earlier inverse agonist DN200434 (Figure 1b),⁵¹ DN201000 exhibited markedly enhanced binding affinity for ERRγ, with an IC₅₀ of 0.035 μM compared to 0.110 μM for GSK5182.⁴³ Crystallographic analysis revealed that DN201000 stabilizes the ERRγ-LBD through hydrogen bonds with Tyr326 and Asn346, π-stacking with His434, and electrostatic interactions with Asp273 (Figure 7f). Beyond improved target engagement, DN201000 demonstrated favorable pharmacokinetic (PK) properties, including higher metabolic stability across liver microsomes and enhanced permeability (1.29 × 10^{− 6} cm/s in PAMPA assays), alongside reduced CYP inhibition (53%, 24%, and 34% at 10 μM for select isoforms) and lower plasma clearance (37 ± 7.7 mL/min/kg) in rats compared to GSK5182.

In cellular studies, DN201000 restored NIS function in ATC cells, inducing maximum radioiodine uptake in CAL62 and BHT101 cell lines at concentrations of 6 and 12 μM, respectively. This effect is correlated with dose- and time-dependent upregulation of iodide-handling genes (TSHR, TPO, TG) at mRNA and protein levels, sensitizing ATC cells to ¹³¹I cytotoxicity. In vivo, oral administration of DN201000 (200 mg/kg) enhanced radioiodine avidity in CAL62 xenografts, as confirmed by ¹²⁴I-PET/CT imaging. These findings position DN201000 as a promising therapeutic candidate for redifferentiation therapy in radioiodine-refractory thyroid cancers.

Pharmacokinetics of Advanced ERRγ Inhibitors.

Kim et al.⁵⁴ discovered an orally available ERRγ inverse agonist compound 25 (Figure 1b), which exhibits enhanced pharmacokinetic properties compared to the previous reported DN201000.⁵³ Compound 25 demonstrated a binding affinity of IC₅₀ = 0.056 μM for ERRγ, comparable to DN201000, while achieving superior bioavailability (45.3%) and an extended plasma half-life (3.0 ± 0.5 h) in rats. The X-ray crystallographic structure of compound 25 revealed a binding motif similar to that of DN201000, with key interactions including hydrogen bonds with Try326, Asn346, and Glu275, electrostatic interactions with Asp273, and π-π stacking with His434 and Phe435 (Figure 7g).

In vitro screening of 30 analogs prioritized compound 25 based on its balanced ADMET (adsorption, distribution, metabolism, excretion, and toxicity) profile, including moderate cytochrome P450 (CYP) enzyme (2C19) inhibition (68% at 10 μM), high permeability (0.84 × 10^{− 6} cm/s in PAMPA), and metabolic stability across species. Pharmacokinetic studies in rats revealed a higher clearance rate (69 ± 7.7 mL/min/kg) and volume of distribution (11.70 ± 4.9 L/kg) relative to DN201000,⁵³ supporting its suitability for oral administration.

Functionally, compound 25 induced redifferentiation in ATC and poorly differentiated thyroid cancer (PDTC) cells by restoring iodide-handling gene expression (NIS, TSHR, TPO) at mRNA and protein levels. Notably, it promoted membrane localization of fully glycosylated NIS (95 kDa), critical for radioiodine uptake. Mechanistically, compound 25 upregulated p44/p42 MAP kinase phosphorylation, linking ERRγ inhibition to MAP kinase-mediated transcriptional activation of iodide-metabolizing genes.

In PDTC (BHP10–3SCp) cells, compound 25 enhanced ¹²⁵I uptake dose-dependently, an effect abolished by the NIS inhibitor KClO₄. Synergy with ¹³¹I irradiation in clonogenic assays reduced colony formation by > 80%, underscoring it potential to resensitize tumors to radioiodine therapy. In vivo, oral administration of compound 25 combined with ¹³¹I significantly suppressed tumor growth in PDTC xenografts, outperforming monotherapies.

ERRγ DBD-DNA Complex: DR1 Recognition and Dimerization.

Zhen et al. reported the first crystal structure of the ERRγ DBD in complex with a direct repeat DNA element (DR1) from the Pla2g12b promoter (G12Bpro).⁵⁸ Biochemical assays demonstrated that ERRγ-DBD exists as a monomer in solution but forms a head-to-tail dimer upon binding the DR1 sequence (5′-GAGGACAAAGGTGAAAC-3′), with a binding affinity of approximately 526 nM. Two DBD molecules contact the DNA asymmetrically, where the downstream monomer binds more tightly (ΔG ≈ − 11.1 kJ/mol) than the upstream one (ΔG ≈ − 6.8 kJ/mol). Sequence-specific recognition is mainly mediated by the first three base pairs of the DR1 motif, and ERRγ shows a clear preference for “G” at the third base position, distinct from HNF4α, which tolerates both “G” and “T.”

The X-ray crystallographic structure revealed that ERRγ binds to the DR1 element as a homodimer (Figure 7h). A key structural feature is a pronounced conformational rearrangement of the CTE in the downstream DBD monomer. In contrast to the canonical binding mode observed in the monomeric ERRβ-ERRE complex,⁴⁰ where the CTE’s A-box folds back into the DNA minor groove, the ERRγ dimer adopts a rearranged CTE conformation that projects away from the DNA (Figure 7i). This repositioning is critical for dimerization, as residues from the upstream monomer, notably Lys178, occupy the minor groove.

This study also identified a distinctive dimer interface mediated by an unconventional T-box conformation. The T-box of the downstream ERRγ molecule is positioned closer to the zinc finger II of the upstream monomer than in other NR-DR1 complexes, establishing a network of specific hydrogen bonds, including interactions between Asp201 and Arg177/Lys178/Ser179 (Figure 7j). The functional significance of this interface was demonstrated using a phosphomimetic mutant (S179E), which mimics a known regulatory modification and markedly decreases DNA binding affinity, increasing the k_d to 0.8 μM. These findings indicate that the T-box conformation not only stabilizes the dimer but also serves as a potential regulatory site.

In addition to the full-length ERRγ DBD structures discussed above, several X-ray structures have been reported in which a short phosphorylated ERRγ DBD peptide (~10 residues) is bound to the 14–3–3 protein.^76,77 While these structures provide useful insight into the recognition of ERRγ phosphopeptides recognition, they do not represent the ERRγ protein itself and are therefore not included in this review.

CONCLUSION AND PERSPECTIVES

The structural characterization of ERRs has fundamentally reshaped our understanding of their ligand-independent activity, coactivator recruitment, and isoform-specific functional diversity. X-ray crystallographic studies have revealed that ERRs adopt unique conformations in their LBDs, enabling constitutive transcriptional activity through interactions with coactivators like PGC-1α. Key structural motifs, such as the hydrophobic LBP and the dynamic H12, govern receptor activation and inhibition. For instance, the repositioning of Phe328 in ERRα and Phe435 in ERRγ upon inverse agonist binding underscores the divergent mechanisms of transcriptional repression across isoforms. These insights have clarified why synthetic ligands like GSK4716 and DN200434 exhibit isoform selectivity, leveraging hydrogen-bond networks (e.g., ERRγ’s Tyr326-Asn346 hydrogen bond).

Therapeutic targeting of ERRs holds immense potential, as evidenced by preclinical successes in metabolic disorders, neurodegenerative diseases, and cancer. Inverse agonists such as DN200434 restore radioiodine avidity in anaplastic thyroid cancer by redifferentiating tumor cells, while environmental ligands like bisphenol analogs highlight the need to address unintended ERRγ activation in disease contexts. However, challenges persist in reconciling the plasticity of ERR LBPs with the design of isoform-selective drugs. The discovery of secondary pockets and the allosteric communication between homodimer interfaces and coactivator-binding sites in ERRα exemplify the complexity of these receptors.⁶⁶ Recent work from our groups has made significant strides toward addressing these challenges. Notably, we have discovered novel modulators of ERRs with demonstrated isoform selectivity and extensive functional validation.^65,78–81 Our development of ERR-targeting compounds incorporates structure-based drug design, high-throughput screening, and functional characterization across cellular and animal models, reflecting a comprehensive and translational approach to ERR pharmacology.^{65,66,78–81} Future priorities should thus focus on compounds that exploit these structural nuances while minimizing off-target effects, particularly given ERRs’ overlapping roles in metabolic and neoplastic pathways.

Advancing ERR pharmacology will require integrating structural biology with computational drug design to overcome current limitations. Molecular dynamics simulations have been increasingly applied to explore ERR conformational dynamics and ligand-induced rearrangements. Several MD studies have reported disturbance of H12 upon binding of inverse agonists, highlighting the intrinsic flexibility of the AF-2 region and its central role in modulating ERR activity.^82–84 Notably, these simulation results are consistent with experimental observation from X-ray crystallographic structures, which also capture H12 repositioning in inverse agonist-bound states. Such findings reinforce the importance of considering dynamic equilibria between active and inactive states when interpreting static crystal structure. MD simulations can further aid in predicting ligands’ binding motif,⁶⁶ while machine learning algorithms might identify novel chemotypes by mining chemical libraries for ERR-selective scaffolds.⁸³ Virtual screening campaigns, guided by high-resolution ERR structures, could lead to the discovery of allosteric modulators or disruptors of pathogenic ERR-coactivator interactions.⁸³ Recent advances of structureprediction algorithms, such as AlphaFold-Multimer and Rosetta, enable modeling of full ERR-coactivator assemblies. While such predictions remain limited by the intrinsically disordered nature of coactivators like PGC-1α, these computational approaches can nevertheless provide complementary insights into the ERR-coactivator interactions.

Structural studies of ERR isoforms have significantly advanced our understanding of their unique ligand-binding properties, constitutive activity, and interaction with coregulators. Through detailed examination of over 40 X-ray crystallographic and NMR structures, this review reveals how specific amino acid differences across ERRα, ERRβ, and ERRγ define ligand selectivity, activation mechanisms, and functional outcomes. Importantly, the discovery and structural validation of ERR-targeting ligands, including agonists, antagonists, and inverse agonists, demonstrate their translational potential in treating metabolic and endocrine disorders, neurodegenerative diseases, and cancers. The integration of crystallographic data with biophysical, cellular, and in vivo functional assays supports the strategic development of ERR-targeted therapeutics. Future research should focus on resolving full-length receptor structures, understanding isoform-specific allosteric regulation, and optimizing ligand selectivity to maximize therapeutic benefit while minimizing off-target effects.

ABBREVIATIONS

AF-2

activation function-2

AhR

aryl hydrocarbon receptor

AMD

age-related macular degeneration

ATC

anaplastic thyroid cancer

AUC

area under the curve

BPB

bisphenol B

BPA

bisphenol A

BPE

bisphenol E

CD

circular dichroism

CNS

central nervous system

CoA

coactivator

CTE

C-terminal extension

CYP

cytochrome P450

DBD

DNA-binding domain

DES

diethylstilbestrol

DR0

direct repeat with zero-base spacing

DR1

direct repeat with one-base spacing

DSC

differential scanning calorimetry

E2

17β-estradiol

EC50

half-maximal effective concentration

EDC

endocrine-disrupting chemical

EMSAs

electrophoretic mobility shift assays

ER

estrogen receptor

ERR

estrogen-related receptor

ERE

estrogen response element

FMO

fragment molecular orbital

FRET

fluorescence resonance energy transfer

H1–H12

helices 1–12

HPLC

high-performance liquid chromatography

HTRF

homogeneous time-resolved fluorescence

IC₅₀

half-maximal inhibitory concentration

IHC

immunohistochemistry

ITC

isothermal titration calorimetry

LBD

ligand-binding domain

LBP

ligand-binding pocket

MAPK

mitogen-activated protein kinase

MD

molecular dynamics

MS

mass spectrometry

NIS

sodium iodide symporter

NMR

nuclear magnetic resonance

NR

nuclear receptor

PDTC

poorly differentiated thyroid cancer

PGC-1α

peroxisome proliferator-activated receptor gamma coactivator 1^α

PGC-1β

peroxisome proliferator-activated receptor gamma coactivator 1β

PK

pharmacokinetics

PPAR

peroxisome proliferator-activated receptor

RAL

raloxifene

RIP140

receptor-interacting protein 140

SAR

structure–activity relationship

SERMs

selective estrogen receptor modulators

SMRT

silencing mediator for retinoid and thyroid hormone receptors

SRC

steroid receptor coactivator

TCA

tricarboxylic acid cycle

TM

melting temperature

TZD

thiazolidinedione

WT

wild-type

4-OHT

4-hydroxytamoxifen

6NI

6-nitroindole