Structural analysis identifies an escape route from the adverse lipogenic effects of liver X receptor ligands.

Liver X receptors (LXRs) are attractive drug targets for cardiovascular disease treatment due to their role in regulating cholesterol homeostasis and immunity. The anti-atherogenic properties of LXRs have prompted development of synthetic ligands, but these cause major adverse effects-such as increased lipogenesis-which are challenging to dissect from their beneficial activities. Here we show that LXR compounds displaying diverse functional responses in animal models induce distinct receptor conformations. Combination of hydrogen/deuterium exchange mass spectrometry and multivariate analysis allowed identification of LXR regions differentially correlating with anti-atherogenic and lipogenic activities of ligands. We show that lipogenic compounds stabilize active states of LXRα and LXRß while the anti-atherogenic expression of the cholesterol transporter ABCA1 is associated with the ligand-induced stabilization of LXRα helix 3. Our data indicates that avoiding ligand interaction with the activation helix 12 while engaging helix 3 may provide directions for development of ligands with improved therapeutic profiles.

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells.

PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Interplay of Protein Disorder in Retinoic Acid Receptor Heterodimer and Its Corepressor Regulates Gene Expression.

In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form.

Role of NCoR1 in mitochondrial function and energy metabolism.

Mitochondrial number and shape are constantly changing in response to increased energy demands. The ability to synchronize mitochondrial pathways to respond to energy fluctuations within the cell is a central aspect of mammalian homeostasis. This dynamic process depends on the coordinated activation of transcriptional complexes to promote the expression of genes encoding for mitochondrial proteins. Recent evidence has shown that the nuclear corepressor NCoR1 is an essential metabolic switch which acts on oxidative metabolism signaling. Here, we provide an overview of the emerging role of NCoR1 in the transcriptional control of energy metabolism. The identification and characterization of NCoR1 as a central, evolutionary conserved player in mitochondrial function have revealed a novel layer of metabolic control. Defining the precise mechanisms by which NCoR1 acts on energy homeostasis will ultimately contribute towards the development of novel therapies for the treatment of metabolic diseases such as obesity and type 2 diabetes.

Binding mode prediction and MD/MMPBSA-based free energy ranking for agonists of REV-ERBα/NCoR.

The knowledge of the free energy of binding of small molecules to a macromolecular target is crucial in drug design as is the ability to predict the functional consequences of binding. We highlight how a molecular dynamics (MD)-based approach can be used to predict the free energy of small molecules, and to provide priorities for the synthesis and the validation via in vitro tests. Here, we study the dynamics and energetics of the nuclear receptor REV-ERBα with its co-repressor NCoR and 35 novel agonists. Our in silico approach combines molecular docking, molecular dynamics (MD), solvent-accessible surface area (SASA) and molecular mechanics poisson boltzmann surface area (MMPBSA) calculations. While docking yielded initial hints on the binding modes, their stability was assessed by MD. The SASA calculations revealed that the presence of the ligand led to a higher exposure of hydrophobic REV-ERB residues for NCoR recruitment. MMPBSA was very successful in ranking ligands by potency in a retrospective and prospective manner. Particularly, the prospective MMPBSA ranking-based validations for four compounds, three predicted to be active and one weakly active, were confirmed experimentally.

The heme-regulatory motif of nuclear receptor Rev-erbß is a key mediator of heme and redox signaling in circadian rhythm maintenance and metabolism.

Rev-erbß is a heme-responsive transcription factor that regulates genes involved in circadian rhythm maintenance and metabolism, effectively bridging these critical cellular processes. Heme binding to Rev-erbß indirectly facilitates its interaction with the nuclear receptor co-repressor (NCoR1), resulting in repression of Rev-erbß target genes. Fe3+-heme binds in a 6-coordinate complex with axial His and Cys ligands, the latter provided by a heme-regulatory motif (HRM). Rev-erbß was thought to be a heme sensor based on a weak Kd value for the Rev-erbß·heme complex of 2 µm determined with isothermal titration calorimetry. However, our group demonstrated with UV-visible difference titrations that the Kd value is in the low nanomolar range, and the Fe3+-heme off-rate is on the order of 10-6 s-1 making Rev-erbß ineffective as a sensor of Fe3+-heme. In this study, we dissected the kinetics of heme binding to Rev-erbß and provided a Kd for Fe3+-heme of ∼0.1 nm Loss of the HRM axial thiolate via redox processes, including oxidation to a disulfide with a neighboring cysteine or dissociation upon reduction of Fe3+- to Fe2+-heme, decreased binding affinity by >20-fold. Furthermore, as measured in a co-immunoprecipitation assay, substitution of the His or Cys heme ligands in Rev-erbß was accompanied by a significant loss of NCoR1 binding. These results demonstrate the importance of the Rev-erbß HRM in regulating interactions with heme and NCoR1 and advance our understanding of how signaling through HRMs affects the major cellular processes of circadian rhythm maintenance and metabolism.

Structure of the MeCP2-TBLR1 complex reveals a molecular basis for Rett syndrome and related disorders.

Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. The majority of RTT missense mutations disrupt the interaction of the MeCP2 with DNA or the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid receptors (SMRT) corepressor complex. Here, we show that the "NCoR/SMRT interaction domain" (NID) of MeCP2 directly contacts transducin beta-like 1 (TBL1) and TBL1 related (TBLR1), two paralogs that are core components of NCoR/SMRT. We determine the cocrystal structure of the MeCP2 NID in complex with the WD40 domain of TBLR1 and confirm by in vitro and ex vivo assays that mutation of interacting residues of TBLR1 and TBL1 disrupts binding to MeCP2. Strikingly, the four MeCP2-NID residues mutated in RTT are those residues that make the most extensive contacts with TBLR1. Moreover, missense mutations in the gene for TBLR1 that are associated with intellectual disability also prevent MeCP2 binding. Our study therefore reveals the molecular basis of an interaction that is crucial for optimal brain function.

Characterization of the Free State Ensemble of the CoRNR Box Motif by Molecular Dynamics Simulations.

Intrinsically disordered proteins (IDPs) and regions are highly prevalent in eukaryotic proteomes, and like folded proteins, they perform essential biological functions. Interaction sites in folded proteins are generally formed by tertiary structures, whereas IDPs use short segments called linear motifs (LMs). Despite their short length and lack of stable structure, LMs may have considerable structural propensities, which often resemble bound-state conformations with targets. Structural data is crucial for understanding the molecular basis of protein interactions and development of targeted pharmaceuticals, but IDPs present considerable challenges to experimental techniques. As a result, IDPs are largely underrepresented in the Protein Data Bank. In the face of experimental challenges, molecular dynamics (MD) simulations have proven to be a useful tool for structural characterization of IDPs. Here, the free state ensemble of the nuclear receptor corepressor 1 (NCOR1) CoRNR box 3 motif, which is important for binding to nuclear receptors to control gene expression, is studied using MD simulations of a total of 8 µs. Transitions between disordered and α-helical conformations resembling a bound-state structure were observed throughout the trajectory, indicating that the motif may have a natural conformational bias toward bound-state structures. The data shows that the disordered and folded populations are separated by a low energy (4-6 kJ/mol) barrier, and the presence of off-pathway intermediates, leading to a C-terminally folded species that cannot efficiently transition into a completely folded conformation. Structural transitions and folding pathways within the free state ensemble were well-described by principal component analysis (PCA) of the peptide backbone dihedral angles, with the analysis providing insight for increasing structural homogeneity of the ensemble.

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbß Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor.

Rev-erbα and Rev-erbß are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbß (FLRev-erbß) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbß·DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbß·NCoR1 complex. The interaction between FLRev-erbß and NCoR1 as well as Rev-erbß repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erbß in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10(-6) s(-1)) of heme dissociation from Rev-erbß rules out a prior proposal that Rev-erbß acts as an intracellular heme sensor.

Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors.

Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment.

Molecular mechanisms of transcriptional control by Rev-erbα: An energetic foundation for reconciling structure and binding with biological function.

Rev-erbα and ß are nuclear receptors that function as transcriptional repressors of genes involved in regulating circadian rhythms, glucose, and cholesterol metabolism and the inflammatory response. Given these key functions, Rev-erbs are important drug targets for treatment of a number of human pathologies, including cancer, heart disease, and type II diabetes. Transcriptional repression by the Rev-erbs involves direct competition with transcriptional activators for target sites, but also recruitment by the Rev-erbs of the NCoR corepressor protein. Interestingly, Rev-erbs do not appear to interact functionally with a very similar corepressor, Smrt. Transcriptional repression by Rev-erbs is thought to occur in response to the binding of heme, although structural, and ligand binding studies in vitro show that heme and corepressor binding are antagonistic. We carried out systematic studies of the ligand and corepressor interactions to address the molecular basis for corepressor specificity and the energetic consequences of ligand binding using a variety of biophysical approaches. Highly quantitative fluorescence anisotropy assays in competition mode revealed that the Rev-erb specificity for the NCoR corepressor lies in the first two residues of the ß-strand in Interaction Domain 1 of NCoR. Our studies confirmed and quantitated the strong antagonism of heme and corepressor binding and significant stabilization of the corepressor complex by a synthetic ligand in vitro. We propose a model which reconciles the contradictory observations concerning the effects of heme binding in vitro and in live cells.

Holocarboxylase synthetase interacts physically with nuclear receptor co-repressor, histone deacetylase 1 and a novel splicing variant of histone deacetylase 1 to repress repeats.

HLCS (holocarboxylase synthetase) is a nuclear protein that catalyses the binding of biotin to distinct lysine residues in chromatin proteins. HLCS-dependent epigenetic marks are over-represented in repressed genomic loci, particularly in repeats. Evidence is mounting that HLCS is a member of a multi-protein gene repression complex, which determines its localization in chromatin. In the present study we tested the hypothesis that HLCS interacts physically with N-CoR (nuclear receptor co-repressor) and HDAC1 (histone deacetylase 1), thereby contributing toward the removal of H3K9ac (Lys9-acetylated histone H3) gene activation marks and the repression of repeats. Physical interactions between HLCS and N-CoR, HDAC1 and a novel splicing variant of HDAC1 were confirmed by co-immunoprecipitation, limited proteolysis and split luciferase complementation assays. When HLCS was overexpressed, the abundance of H3K9ac marks decreased by 50% and 68% in LTRs (long terminal repeats) 15 and 22 respectively in HEK (human embryonic kidney)-293 cells compared with the controls. This loss of H3K9ac marks was linked with an 83% decrease in mRNA coding for LTRs. Similar patterns were seen in pericentromeric alpha satellite repeats in chromosomes 1 and 4. We conclude that interactions of HLCS with N-CoR and HDACs contribute towards the transcriptional repression of repeats, presumably increasing genome stability.

Profiling of substrates for zinc-dependent lysine deacylase enzymes: HDAC3 exhibits decrotonylase activity in vitro.

Systematic screening of the activities of the eleven human zinc-dependent lysine deacylases against a series of fluorogenic substrates as well as kinetic evaluation revealed substrates for screenings of histone deacetylases HDAC10 and HDAC11 at reasonably low enzyme concentrations. Furthermore, HDAC3 in complex with nuclear receptor corepressor 1 (HDAC3-NCoR1) was shown to harbor decrotonylase activity in vitro.

Resistance to thyroid hormone is modulated in vivo by the nuclear receptor corepressor (NCOR1).

Mutations in the ligand-binding domain of the thyroid hormone receptor ß (TRß) lead to resistance to thyroid hormone (RTH). These TRß mutants function in a dominant-negative fashion to interfere with the transcription activity of wild-type thyroid hormone receptors (TRs), leading to dysregulation of the pituitary-thyroid axis and resistance in peripheral tissues. The molecular mechanism by which TRß mutants cause RTH has been postulated to be an inability of the mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone (TH)-mediated transcription activity. To test this hypothesis in vivo, we crossed Thrb(PV) mice (a model of RTH) expressing a human TRß mutant (PV) with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit a TR or a PV mutant. Remarkably, in the presence of NCOR1ΔID, the abnormally elevated thyroid-stimulating hormone and TH levels found in Thrb(PV) mice were modestly but significantly corrected. Furthermore, thyroid hyperplasia, weight loss, and other hallmarks of RTH were also partially reverted in mice expressing NCOR1ΔID. Taken together, these data suggest that the aberrant recruitment of NCOR1 by RTH TRß mutants leads to clinical RTH in humans. The present study suggests that therapies aimed at the TR-NCOR1 interaction or its downstream actions could be tested as potential targets in treating RTH.

GSK4112, a small molecule chemical probe for the cell biology of the nuclear heme receptor Rev-erbα.

The identification of nonporphyrin ligands for the orphan nuclear receptor Rev-erbα will enable studies of its role as a heme sensor and regulator of metabolic and circadian signaling. We describe the development of a biochemical assay measuring the interaction between Rev-erbα and a peptide from the nuclear receptor corepressor-1 (NCoR). The assay was utilized to identify a small molecule ligand for Rev-erbα, GSK4112 (1), that was competitive with heme. In cells, 1 profiled as a Rev-erbα agonist in cells to inhibit expression of the circadian target gene bmal1. In addition, 1 repressed the expression of gluconeogenic genes in liver cells and reduced glucose output in primary hepatocytes. Therefore, 1 is useful as a chemical tool to probe the function of Rev-erbα in transcriptional repression, regulation of circadian biology, and metabolic pathways. Additionally, 1 may serve as a starting point for design of Rev-erbα chemical probes with in vivo pharmacological activity.

Interplay of protein misfolding pathway and unfolded-protein response in acute promyelocytic leukemia.

Protein misfolding has traditionally been linked to the pathogenesis of various neurodegenerative diseases. However, emerging evidence from various laboratories, including ours, suggests that protein misfolding may also play a fundamental role in some malignancies, particularly those caused by fusion oncoprotein generated from chromosomal translocation. Promyelocytic leukemia (PML) fused to the retinoic acid receptor (RAR) is a fusion oncoprotein linked to the transformation of acute promyelocytic leukemia (APL), and is not only a misfolded protein itself, but also promotes misfolding of nuclear receptor corepressor (N-CoR) protein, a corepressor essential for the growth-suppressive function of several tumor-suppressor proteins. PML-RAR promotes misfolding of N-CoR by inducing aberrant post-translational modification, which destabilizes its core and promotes instability. Misfolded N-CoR, thus, contributes to differentiation arrest and survival of APL cells through loss-of-function and aberrant gain-of-function properties. Therapeutic restoration of N-CoR conformation and function with conformation-modifying agents not only releases this differentiation arrest but also sensitizes APL cells to programmed cell death. These findings illustrate the potential of the misfolded N-CoR protein as a conformation-based drugable molecular target for APL, and highlights the promise of various conformation-modifying agents as novel therapeutics for APL. Protein conformational rearrangement, resulting from an inherited or acquired genetic alteration, could be a common pathological phenomenon contributing to transformation in different types of leukemias and solid tumors and, therefore, could serve as a common ground for designing a unifying diagnostic as well as therapeutic approach for a widely diverse disease such as cancer. To that end, APL could serve as a model for the development of a novel conformation-based therapeutic approach for other malignant diseases.

Structure of Rev-erbalpha bound to N-CoR reveals a unique mechanism of nuclear receptor-co-repressor interaction.

Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.

A unique secondary-structure switch controls constitutive gene repression by retinoic acid receptor.

In the absence of ligand, some nuclear receptors, including retinoic acid receptor (RAR), act as transcriptional repressors by recruiting corepressor complexes to target genes. This constitutive repression is crucial in metazoan reproduction, development and homeostasis. However, its specific molecular determinants had remained obscure. Using structural, biochemical and cell-based assays, we show that the basal repressive activity of RAR is conferred by an extended beta-strand that forms an antiparallel beta-sheet with specific corepressor residues. Agonist binding induces a beta-strand-to-alpha-helix transition that allows for helix H11 formation, which in turn provokes corepressor release, repositioning of helix H12 and coactivator recruitment. Several lines of evidence suggest that this structural switch could be implicated in the intrinsic repressor function of other nuclear receptors. Finally, we report on the molecular mechanism by which inverse agonists strengthen corepressor interaction and enhance gene silencing by RAR.

Estrogen receptors recruit SMRT and N-CoR corepressors through newly recognized contacts between the corepressor N terminus and the receptor DNA binding domain.

Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.

Molecular determinants required for selective interactions between the thyroid hormone receptor homodimer and the nuclear receptor corepressor N-CoR.

The unliganded nuclear receptor (NR) generally recruits the NR corepressor (N-CoR) and the silencing mediator of retinoid and thyroid hormone receptor via its direct binding to the extended helical motif within dual NR-interaction domains (IDs) of corepressors. Interestingly, N-CoR has a third ID (ID3) upstream of two IDs (ID1 and ID2) and its core motif (IDVII), rather than an extended helical motif, is known to be involved directly in the exclusive interaction of ID3 with the thyroid hormone receptor (TR). Here, we investigated the molecular determinants of the TR interaction with ID3 to understand the molecular basis of the N-CoR preference shown by the TR homodimer. Using a one- plus two-hybrid system, we identified the specific residues of N-CoR-ID2 and N-CoR-ID3 that are required for stable association of N-CoR with the TR homodimer. By swapping experiments and mutagenesis studies, we found that the C-terminally flanked residues of the core motif of ID3 contribute to the TR preference for N-CoR-ID3, suggesting that an extended three-turn helix might form within the ID3 via a C-terminal extension (IDVIITRQI) and participate directly in the TR-specific interaction. Structural modeling of the ID3 motif on TR-LBD is consistent with this conclusion. Notably, we identified a novel interaction between N-CoR-ID3 and orphan NR RevErb that is mediated by the residues crucial also in TR binding. These observations raise the intriguing possibility that NR homodimers such as TR and RevErb display preferential binding to the N-CoR corepressor via their specific interactions with ID3, which is normally absent from the silencing mediator of retinoid and thyroid hormone receptor.