RXR Agonist Modulates TR: Corepressor Dissociation Upon 9-cis Retinoic Acid Treatment

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1 ORIGINAL RESEARCH RXR Agonist Modulates TR: Corepressor Dissociation Upon 9-cis Retinoic Acid Treatment Juliana Fattori, Jéssica L. O. Campos, Tábata R. Doratioto, Lucas M. Assis, Mariela T. Vitorino, Igor Polikarpov, José Xavier-Neto, and Ana Carolina M. Figueira Centro Nacional de Pesquisa em Energia e Materiais (J.F., J.L.O.C., T.R.D., L.M.A., M.T.V., J.X.-N., A.C.M.F.), Laboratório Nacional de Biociências, Campinas SP, , Brazil; and Instituto de Física de São Carlos (I.P.), Universidade de São Paulo, São Carlos SP, , Brazil Transcriptional regulation controlled by thyroid hormone receptor (TR) drives events such as development, differentiation, and metabolism. TRs may act either as homodimers or as heterodimers with retinoid X receptor (RXR). Thyroid hormone T 3 preferentially binds TR-RXR heterodimers, which activate transcription through coactivator recruitment. However, it is unclear whether TR-RXR heterodimers may also be responsive to the canonical RXR agonist 9-cis retinoic acid (9C) in the context of physiological gene regulation. New structural studies suggest that 9C promotes the displacement of bound coactivators from the heterodimer, modifying TR-RXR activity. To shed light on the molecular mechanisms that control TR-RXR function, we used biophysical approaches to characterize coregulator recruitment to TR-TR or to TR-RXR in the presence of T 3 and/or 9C as well as cell-based assays to establish the functional significance of biophysical findings. Using cell-based and fluorescence assays with mutant and wild-type TR, we show that 9C does indeed have a function in the TR-RXR heterodimer context, in which it induces the release of corepressors. Furthermore, we show that 9C does not promote detectable conformational changes in the structure of the TR-RXR heterodimer and does not affect coactivator recruitment. Finally, our data support the view that DNA binding domain and Hinge regions are important to set up NR-coactivator binding interfaces. In summary, we showed that the RXR agonist 9C can regulate TR function through its modulation of corepressor dissociation. (Molecular Endocrinology 29: , 2015) Thyroid hormone receptor (TR) is a nuclear receptor (NR) responsible for the regulation of basal metabolism, metamorphosis, growth, and other developmental functions (1). As transcription factors modulated by small lipophilic molecules, TRs control the expression of target genes involved in diverse physiological processes and diseases, such as metabolic syndrome, obesity, and cancer, and, therefore, are considered as important targets for therapeutic drug development (2). ISSN Print ISSN Online Printed in U.S.A. Copyright 2015 by the Endocrine Society Received August 8, Accepted December 19, First Published Online December 26, 2014 It is known that TRs may act as homodimers, or heterodimers with retinoid X receptor (RXR), to induce or repress gene expression (3 11). The heterodimer TR- RXR is the most active complex for gene regulation (4). Classically, in the absence of ligand, TR is able to recruit corepressors, which leads to gene silencing. In the presence of ligand, TR undergoes changes in its conformation and promotes coactivator recruitment, which activates transcriptional complexes and lead to gene activation (12, Abbreviations: AUC, analytical ultracentrifugation; 9C, 9-cis retinoic acid; CD, circular dichroism; CoA, coactivator; DBD, DNA binding domain; DLS, dynamic light scattering; DR4, direct repeat spaced by 4 nucleotides response element; DSF, differential scanning fluorimetry; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; GRIP1, glucocorticoid receptor interacting protein 1; H, helix; h, human; HEK, human embryonic kidney; K d, affinity constant; LBD, ligand binding domain; Mr, molecular mass; N-Cor, NR corepressor; NR, nuclear receptor; R h, hydrodynamic radii; RID, receptor interaction domain; RXR, retinoid X receptor; SAXS, small-angle X-ray scattering; SEC, size exclusion chromatography; SRC-1, steroid receptor coactivator-1; Tm, temperature midpoint of the protein unfolding transition; TR, thyroid hormone receptor. 258 mend.endojournals.org Mol Endocrinol, February 2015, 29(2): doi: /me

2 doi: /me mend.endojournals.org ). Although this classical mechanism remains a useful first approximation, signal transduction in the NR signaling pathway is much more complex and requires a host of interactions among NRs, coactivators, corepressors, and the transcriptional machinery (14, 15). One of the still unclear areas concerning NR-dependent signaling is highlighted by physiological situations in which the gene regulation by NRs also requires ligandbound receptors to repress gene transcription (14). Therefore, it is important to understand the molecular basis of ligand-induced repression and how it differs from ligandinduced activation. As most NRs, TRs share a multidomain structure, which contains DNA binding and ligand binding domains (DBD and LBD, respectively). DBD is crucial for interaction with DNA regulatory sequences, whereas the LBD displays the main dimerization interface for homo- and heterodimerization (16). The LBD is responsible for most of the conformational changes after ligand binding, chiefly by relocating of its helix (H) 12 after ligand binding. This structural rearrangement generates the major TR-coactivator (CoA) binding interface, which generally is formed between motifs LXXLL present in most coactivators [receptor interaction domain (RID)] and in the hydrophobic surface created by helixes H3, H4, H5, and H12 of the TR LBD (16). Beyond the above-mentioned universally accepted model, the physiological interactions between NR and coregulators are not fully understood, partially because structural and biophysical data from NR-coactivator complexes have been, so far, restricted to minimal domains, such as truncated LBDs and LXXLL peptides (16). Although more than 500 deposited NR crystal structures have provided invaluable structural information on the interactions between LBDs and LXXLL motifs (Protein data banking, only recently full-length NR structures have begun to reveal higher-order quaternary organization (17 20). In general, these novel and more complete structures indicate the importance of interdomain interaction in NR regulation and suggest that there are still undisclosed interactions to be characterized. More specifically, some of these studies suggest additional interfaces between NRs and coregulators, which sometimes bind to the N-terminal portion of NRs and, sometimes, bind to the DBD (21 24). Therefore, it is likely that studies with full length NR and coregulators will reveal potentially new and relevant specific contacts between NR and coregulators, which may dictate the rules of complex formation underlying the sophisticated regulation of physiological processes by these transcription factors. Another subject that is still not well defined in NR regulation is heterodimer permissiveness. It is known that some of the RXR heterodimers, such as peroxisome proliferator-activated receptor-rxr, retinoic acid receptor- RXR, vitamin D receptor-rxr, are permissive to 9-cis retinoic acid (9C) binding by RXR (25 27). Initially, TR- RXR was considered nonpermissive because it is unresponsive to RXR agonists alone (25, 28, 29). Although this evidence points to RXR as a silent partner of TR (27, 30), it was suggested that binding of 9C to RXR may display a modulator role on TR activity (30). Consistent with this, 9C binding to TR-RXR heterodimer has been reported to induce allosteric changes on TR, which lead to T 3 dissociation (31). Moreover, studies on Xenopus laevis development indicated that RXR is critical for TR function (32). Additionally, it was suggested that 9C may play regulatory roles on TR-RXR activity by decreasing coactivator affinity (33) or by improving coactivator recruitment, depending on the cellular context (31, 34). Finally, functional evidence suggests that 9C modulates gene repression by TR through dissociation of the corepressor complexes from TR-RXR heterodimers (35) and that the RXR N-terminal domain and DBD are important for corepressor interaction and inhibit TR activity (36). To acquire insights on some still unsolved questions about TR-RXR regulation, in this study we investigated the following: 1) whether 9C actually binds the TR-RXR heterodimer; 2) whether TR-RXR is permissive to 9C; 3) whether 9C provokes structural changes in the TR-RXR heterodimer, 4) whether 9C modifies corepressor or coactivator recruitment/dissociation; 5) whether there is any preference in the complex formation between the coactivator and TR homo- or heterodimers; and 6) whether the DBD is important for complex stabilization. Here we show in biophysical assays that 9C does bind the TR- RXR complex and that it increases the activation of a thyroid reporter in the context of human embryonic kidney (HEK)-293T cells transfected with the glucocorticoid receptor interacting protein 1 (GRIP1) coactivator. Using a mutational approach, we determined that 9C increases reporter activity by inducing corepressor [NR corepressor (N-Cor)] dissociation. In summary, our data indicates that the TR-RXR heterodimer is permissible to 9C, which plays a role in TR function through modulation of corepressor dissociation. Materials and Methods Protein expression and purification Expression and purification of NRs NRs human (h) TR 1 LBD (amino acids ), htr 1 AB (amino acids ), hrxr -LBD (amino acids , expression vector pet-17), and hrxr AB (amino

3 260 Fattori et al RXR Agonist Modulates Derepression of TR Mol Endocrinol, February 2015, 29(2): acids ) were expressed using pet28a( ) expression vector in BL21(DE3) Escherichia coli cells. Bacteria harboring the expression plasmid were grown in Luria broth medium at 22 C, and protein expression was induced with 0.5 mmol/l 1 isopropylthio- -D-galactoside (16 h). Cells were harvested by centrifugation at 4 C. The pellet was resuspended in 20 mmol/ L 1 HEPES (ph 8), 300 mmol/l 1 NaCl, and 5% glycerol containing 2 mol/l 1 of -mercaptoetanol and 100 mol/l 1 phenylmethylsulfonylfluoride, and lysozyme was added to the suspension and extract was sonicated and centrifuged for clarification. Proteins were purified by with Talon resin (CLONTECH) and were eluted with 20 mmol/l 1 HEPES (ph 8), 300 mmol/ L 1 NaCl, 5% glycerol, 300 mmol/l 1 imidazole, and 2 mol/ L 1 of -mercaptoethanol. Purity of samples was evaluated by SDS-PAGE. Expression and purification of coactivator GRIP1 The coactivator MmSRC-2 or GRIP1 (amino acids ) was expressed in pgex-2t. BL21(DE3) E coli cells containing the expression plasmid were grown in Luria broth medium at 22 C, and protein expression was induced with 0.5 mmol/l 1 isopropylthio- -D-galactoside (16 h). Cells were harvested and the pellet was resuspended in PBS buffer containing 100 mol/l 1 phenylmethylsulfonylfluoride and lysozyme. The suspension was sonicated and centrifuged for clarification. GRIP1 was eluted from glutathione-sepharose 4 fast flow (Amershan Biosciences) resin using PBS buffer supplemented with 5 mmol/l 1 reduced glutathione and 1 mmol/l 1 dithiothreitol (DTT). Purity of samples was evaluated by SDS-PAGE. Complex formation Initially the coactivator GRIP1 freshly purified was incubated with glutathione-sepharose 4 fast flow (Amershan Biosciences) resin. Purified receptors TR T 3 and TR T 3 -RXR (T 3 was obtained from Sigma-Aldrich) were added to GRIP1-resin suspension after an incubation at 4 C. In the sequence, thrombin was added to the suspension, and the cleavage was performed for 16 hours, at 4 C, with stirring. After cleavage the collected flow-through presents the complex NRs-CoA. The complex presence was confirmed by SDS-PAGE and native page. Biophysical characterization Analytical gel filtration experiments were executed using a Superdex /300 GL column (GE Healthcare Life Sciences) at 4 C following the manufacturer s protocols. The chromatography buffer was 20 mmol/l 1 Tris (ph 8.0), 300 mmol/l 1 NaCl, and glycerol 5% and 1 mmol/l 1 DTT. Globular proteins with known hydrodynamic radii (R h ), obtained from GE Healthcare Life Sciences, were used as standards to calibrate the column: ferritin [molecular mass (Mr) Da, R h 61.0 Å], aldolase (Mr Da, R h 48.1 Å), conalbumin (Mr Da, R h 36.0 Å), ovalbumin (Mr Da, R h 30.5 Å), carbonic anydrase (Mr Da, R h 20.0 Å), and ribonuclease (Mr Da, R h 16.4 Å). Molecular mass calculations and hydrodynamic stokes radii were determined using least squares fit of gel phase distribution coefficient (K av )vslog molecular weight and (logk av ) 1/2 vs R h, respectively (37). Far-UV circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter with peltier control using 1 mm Quartz cuvettes (Hellma). CD spectra were recorded from 260 to 190 nm at 10 C with a scan speed of 20 nm/min, 20 accumulations, and a response time of 4 seconds. A protein concentration of 2 5 mol/l 1 was used in 5 mmol/l 1 Tris buffer (ph 8.0) containing 25 mmol/l 1 NaCl. Equivalent spectra of buffers were recorded and subtracted from the proteins spectra. Thermal unfolding were executed monitoring CD signal at 222 and 208 nm from 10 C to 90 C, with a temperature rate of 1 C/min. Data were fitted using Origin software (version 8.0; OriginLab Corp), which applies the Levenberg- Marquardt algorithm, with Boltzmann function for fitting curves to nonlinear equations, and data were converged until they reach the software tolerance criteria, with 400 for the maximum number of iterations. CD spectra were deconvoluted using DichroWeb (38, 39) and experiments were performed in triplicates. Dynamic light scattering (DLS) measurements of all sample solutions were conducted using a Protein Solutions DynaPro DLS system (Wyatt). For each sample, one measurement corresponds to 100 acquisitions of 5 seconds, at 10 C. Differential scanning fluorimetry (DSF) experiments were performed, in triplicates, to evaluate the tertiary stability of the proteins under study. In this assay, we monitored protein thermal unfolding by the increase in the fluorescence of SYPRO Orange probe (Life Technologies). Protein samples (2 mol/ L 1 ) in 20 mmol/l 1 Tris buffer (ph 8.0) containing 500 mmol/ L 1 NaCl, 10% glycerol, and SYPRO Orange in a reaction volume of L were incubated in 96-well PCR microplates (Applied Biosystems-Life Technologies) in the RT-PCR device (7500 real time PCR system). Samples were heated at 1 C/min, from 10 C to 100 C. Fluorescence intensities were plotted as a function of temperature and the temperature midpoint of the protein unfolding transition (Tm) was obtained through a Boltzmann model using an Excel-based worksheet (DSF Analysis version 3.0.2) provided by Niesen et al (40).We also used Origin software (version 8.0; OriginLab Corp) to fit average curves, which applies the Levenberg-Marquardt algorithm, with Boltzmann function and 400 interactions until the fitting converges with a software tolerance criterion. Analytical ultracentrifugation Sedimentation velocity experiments for TR-GRIP1 complex were performed in a BeckmanCoulter Optima XL-A (Beckman Instruments Inc) ultracentrifuge at 10 C, and samples were rotate at rpm with absorbance at 280 nm monitoring. Protein concentrations were in the range of 0.8 to 2.0 mg/ml in a PBS buffer. Consecutive scans were automatically recorded at regular intervals and analyzed with the software Sedfit (41). Small-angle X-ray scattering (SAXS) SAXS data were recorded on SAXS2 beam line at the synchrotron facility LNLS (Brazilian Synchrotron Light Laboratory, Brazil) equipped with a MARCCD detector. The wavelength of the X-rays was 1.54 Å and the sample-to-detector distance was mm. The effective Q range extended from 0.01 Å 1 to 0.3 Å 1. The experimental buffer was 20 mmol/l 1 Tris (ph 8.0), 500 mmol/l 1 NaCl, 10% glycerol, and 1 mmol/ L 1 DTT. Protein samples were prepared in concentrations of 2 and 5 mg/ml in the experimental buffer. The temperature of the sample holder was kept constant at 10 C using a water-bath

4 NR complex stability To monitor protein folding, we used CD and DSF. We established parameters of secondary and tertiary structures for TR, RXR, and for GRIP1 in isolation or in protein complexes. In Figure 1, CD spectra show that all individual proteins and complexes are folded and display the expected content of secondary structures, which are composed mostly by -helices (minimum at 208 and 222 nm), except for GRIP1, which presents itself as a mix of -sheet (31%) and unfolded structural patterns (25% of turns and 33% of unordered structure), as calculated from spectra deconvolution (Supplemental Table 1 and Supplemental Material). Upon formation of complexes with NRs plus GRIP1, we observed a clear decrease in spectral features attributed to misfolded structures (14% 17% of turns and 30% 27% of unordered), which suggests that GRIP1 gains structured features after binddoi: /me mend.endojournals.org 261 temperature controller. Scattering patterns for protein samples and buffers were collected alternatively with exposure times of 300 seconds, optimized to reduce radiation damage. Data reduction included averaging of individual curves with PRIMUS (42). The radius of gyration (Rg) and the scattering intensity at zero angle, I (0), were estimated using the Guinier approximation [I(q) I (0)exp( q 2 Rg 2/3 )], valid for small angles (q 1.3/Rg). GNOM (43) was used to obtain the distance distribution function, p(r), and maximum intramolecular distance. Kratky plots [q 2 I(q) q] calculated from the scattering data were used to assess the conformational state of the proteins in solution (44 46). Because GRIP1 may adopt diverse conformations in solution, we decided not to build SAXS models for proteins and complexes. Fluorescence anisotropy The affinities of TR and TR-RXR for GRIP1 and coregulator peptides, and of TR T 3 -RXR-GRIP1 complex for the direct repeat spaced by 4 nucleotides response element (DR4) were measured by titration assays using fluorescence anisotropy, as previously described (11, 47). Purified GRIP1 was labeled with fluorescein isothiocyanate (FITC) and kept in a cuvette, in 50 nmol/l 1 stock solution. This solution was titrated with TR and TR-RXR solutions, and fluorescence polarization was measured in an ISS PC-1 fluorimeter (ISS), using 495 nm as the excitation wavelength, at 10 C, with five iterations. The same procedure was performed for labeled coregulator peptides steroid receptor coactivator-1 (SRC-1; fluorescein- SRC1 1 coactivator peptide 3 FITC-KYSQTSHKLVQLLTT TAEQQL) and N-Cor (fluorescein-n-cor ID1 corepressor peptide 3 FITC-RTHRLITLADHICQIITQDFARN), both from Life Technologies. However, in this case, experiments were made in two steps. First, SRC-1 peptide solution was titrated with TR T 3 -RXR, and N-Cor peptide was titrated with TR- RXR until it reaches saturation, forming a plateau. In the second step, 9C (9C retinoic acid was obtained from Sigma-Aldrich) was titrated into TR-RXR-peptide solutions. For protein-dna affinities, it was used DR4 labeled with FITC (5 -FITC-AGCTAAAGGTCAGATCAGGTCAGTAGG A-3 -IDT). DNA was diluted to 10 nmol/l 1 in binding buffer (20 mmol/l 1 Tris, ph 8.0; 150 mmol/l 1 NaCl; 2 mm MgCl 2 ; and 1 mm DTT). Protein complex stocks were diluted in the same buffer. DR4 labeled was titrated with TR T 3 -RXR- GRIP1 in the presence and absence of 9C, and the fluorescence anisotropy values were measured, at 15 C, using the same set-up for protein-protein affinities. All fluorescence curves were fit as described (11, 47), using Origin software (version 8.0; OriginLab Corp), which applies the Levenberg-Marquardt algorithm for fitting curves to nonlinear equations, to determine the affinity constant (K d ) and Hill coefficient values. All fluorescence experiments were performed in triplicate. Quenching fluorescence Quenching experiments, also performed in triplicates, were executed by titration of 9C in protein solutions, measuring tryptophan fluorescence in an ISS PC-1 fluorimeter, using 280 nm as the excitation wavelength at 15 C and scanning fluorescence emission from 300 to 400 nm. Protein solutions were at 5 mol/ L 1 in PBS buffer with 2% glycerol (48 50). Data were fit using Origin software (version 8.0; OriginLab Corp). Cell transactivation assays HEK293T cells were maintained in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 50 U/mL penicillin, and 50 mg/ml streptomycin under 95% air-5% CO 2 atmosphere at 37 C. For transfection, cells were trypsinized, resuspended in DMEM, plated in 24-well plates (density of cells/ well), and incubated with the following plasmids: pbluescript (used as empty DNA to equilibrate DNA quantity); prl (which contains Renilla reniformis luciferase) used as the transfection control; DR-4Luc (plasmid that contains responsive element for heterodimer TR-RXR followed by firefly luciferase reporter gene); TR full CMV (plasmid with TR- full gene); RXR full CMV (plasmid with RXR- full gene); GRIP1 (plasmid with coactivator GRIP1 gene); TR F451X (plasmid with TR mutant F451X full gene); and TR V458M (plasmid with TR mutant V458M full gene) (51 55). Plasmids were mixed with Lipofectamine 2000 (Invitrogen) at a ratio of 1.5 g of DNA to 2 L of Lipofectamine, at room temperature, for 20 minutes before addiction to the cells. Ligands, T 3, and 9C were added (1 M) to the culture medium 4 hours after the transfection. The cell monolayer was harvested 48 hours later with lysis buffer (dual luciferase report assay system; Promega) according to the manufacturer s instructions. Luciferase activity was determined using a luciferase assay system (Promega) and measured in Glo- Max-Multi detection system (Promega). The R reniformis luciferase activity was measured using the same cell lysate and used as an internal control. Transfection of the DR4 reporter along with pbluescript and treatments with T 3, 9C, and T 3 9C were performed as a control of each reporter activation assay. In these conditions all luciferase signals are due to endogenous activation of any NR in DR4. The activation values for TR (wild type and mutants), RXR, and conditions that favored TR-RXR heterodimers were obtained by normalization with this control. ANOVA and Bonferroni s post hoc comparison were used for statistical analysis to compare each experimental condition. Values with P.05 was considered statistically significant. Results

5 262 Fattori et al RXR Agonist Modulates Derepression of TR Mol Endocrinol, February 2015, 29(2): Figure 1. CD spectra of TR, RXR, GRIP1, and complexes of truncated forms (short LBD), showing that only GRIP1 has coiled coil -sheet structural pattern, whereas TR and its complexes (TR, TR T 3,TR T 3 -GRIP1, TR T 3 - RXR, TR T 3 -RXR-GRIP1, and TR T 3 -RXR 9C-GRIP1) present secondary structure composed mostly by -helixes (minimum at 208 and 222nm). ing to TR homodimers (TR-TR) or to heterodimers (TR-RXR). To check whether complex formation improved protein stability, we used secondary and tertiary structure stability assays such as a thermal unfold using CD, or DSF. Interestingly, thermal unfold experiments showed that, despite the inherent stability of the globular NR LBD, the TR-RXR- GRIP1 complex formed by the widely used truncated versions of TR and RXR (short LBDs) displayed lower stability than TR-RXR-GRIP1 complexes formed by less truncated forms of RXR and TR (DBDs plus LBDs), which are evaluated by the calculated Tms and their associated errors (Table 1, Supplemental Figure 1, and Supplemental Material). These results suggest that as more complete NR sequences are used, more contacts between TR-RXR and coactivator (GRIP1) are established. The increase in stability between receptor heterodimer and GRIP1 observed by CD in the presence of less truncated versions of TR and RXR (DBD-LBD) was largely confirmed by DSF experiments. DSF data show that melting Tms of tertiary structure loss increase with coactivator binding when we use less truncated NR constructions (DBD-LBD), in contrast to truncated forms (short LBD) (Supplemental Figure 2, Supplemental Material, and Table 1). Overall, CD and DSF data suggest that there are still undisclosed, potentially important protein-protein interactions among NRs and coregulators. 9-C does bind to the TR-RXR heterodimer To establish whether 9C binds to the TR-RXR heterodimer, we used a fluorescence-quenching approach that uses the intrinsic fluorescence of RXR s W305 residue, which is located at the binding site entry and has its emission quenched after 9C binding and H12 closing (31, 56) (Figure 2). In all cases (RXR, TR-RXR, TR T 3 - RXR, and TR T 3 -RXR-GRIP1), we observed 20% 45% suppression of tryptophan fluorescence after 9C titration, indicating that 9C actually binds RXR alone or in complex with TR or TR GRIP1. The major quenching was observed, as expected, for RXR-LBD (45%), with almost no variation of center of spectral mass (Supplemental Figure 3 and Supplemental Material). TR-RXR heterodimers with and without T 3 displayed suppression of approximately 30% of fluorescence intensity, whereas for the TR T 3 -RXR-GRIP1 complex, fluorescence suppression was approximately 20%, with the highest variation in the center of the spectral mass. This quenching indicates that 9C does bind to TR T 3 -RXR-GRIP1 and therefore that the TR-RXR heterodimer might be permissive to 9C binding. 9C binding increases the stability of complexes formed by truncated forms of TR and RXR (LBDs) To investigate whether 9C binding has an impact on the formation of molecular complexes containing liganded or unliganded TR-RXR-GRIP1, we set out to measure its effects on complex stability. Interestingly, we established that the Tms of secondary and tertiary structure increased 7 C and 6 C for complexes in the presence, or in the absence of 9C, respectively, which is considered a significant stabilization, once Tm differences are bigger than the associated standard errors from each calculated Tm (57). This suggests that, when truncated forms of TR and RXR are used, 9C displays a clear stabilizing effect on coactivator (GRIP1) complexes (Table 1). 9C binding does not increase the stability of complexes formed by less truncated forms of TR and RXR (DBDs-LBDs) In contrast to the results obtained with truncated NRs described above (which contain only LBD sequences), our Table 1. Comparison Between the Stability of Secondary and Tertiary Structures (Tm/ C) of TR, TR-RXR, TR-GRIP1, and TR-RXR-GRIP1 in the Presence or Absence of Ligands CD DSF Analysis Protein LBD DBD-LBD LBD DBD-LBD TR T TR T 3 -RXR TR T 3 -GRIP TR T 3 -RXR-GRIP TR T 3 -RXR 9C-GRIP

6 doi: /me mend.endojournals.org 263 Figure 2. Tryptophan fluorescence quenching experiments. A, RXR structure in the absence of ligand (Protein Data Bank identification 1LBD) evidencing tryptophan 305 (sticks) after 9C binding (gray, spheres, Protein Data Bank identification 3UVV). H12 is packed against the body of LBD, which hides this tryptophan, causing fluorescence quenching (56). B, Fluorescence intensity decay of RXR, TR-RXR, TR T 3 -RXR, TR T 3 -RXR-GRIP1 solutions (5 M) upon 9C addition (average curves with error bars). Lysozyme was used as control to verify possible inner filter effects. CD and DSF data indicate that 9C changes neither the profile and content of secondary and tertiary structures nor the stability of less truncated forms of heterodimers, composed by DBD plus LBD (Table 1). This might indicate that the presence of further native DBD structures in the less truncated TR effectively masked the evident stabilizing effects that 9C displayed on the truncated LBD core. Size characterization of complexes by size exclusion chromatography (SEC), DLS, and SAXS Techniques that provide insights on protein stability by taking advantage of thermal changes represent convenient approaches for a rapid assessment of the status of NR complexes. However, these techniques cannot give direct information on the size or on the oligomeric state of these complexes. To verify the sizes of isolated proteins and their complexes, we used analytical SEC, DLS, and SAXS assays. Our SEC results show that in solution TR and RXR are represented by a mix of oligomeric forms in which heterodimers constitute the most prevalent species (Table 2 and Supplemental Figure 4). Upon addition of GRIP1, there is a clear shift to one preferential complex, which, as evaluated by the calculated hydrodynamic radius (R h 4.8 nm), is consistent with one TR homodimer bound to one coactivator molecule or one TR-RXR heterodimer bound to one molecule of the same coactivator. Dynamic light scatterings were made after gel filtration to confirm our complex characterization (Table 2). In these measurements we confirm that TR T 3 samples are presented as a mixture of monomer and dimer in equilibrium favoring to monomer (Supplemental Figure 4 and Supplemental Table 2) (58). Although RXR in presence or absence of 9C is as a mixture of tetramer and monomer in solution, in equilibrium that favors tetramer, as also described (59). The heterodimer, on the other hand, exists in solution with low polidispersity (5.4%) with a hydrodynamic radius of 3.6 Å. This low polidispersity implies in low variation of species in the sample, which presents the majority radii with the same size of dimers. Therefore, the sample low monodispersity and the calculated radio, along with the results shown above helps to support the idea that 9C is binding to RXR in the heterodimer, instead of sequester RXR from the TR T 3 -RXR or TR T 3 -

7 264 Fattori et al RXR Agonist Modulates Derepression of TR Mol Endocrinol, February 2015, 29(2): Table 2. Comparison Between the R h Measured by DLS, SEC, and SAXS LBD Protein DLS, nm SEC, nm SAXS, nm a TR 3.2 (D) 3.6 (D) (D) TR T 3 3 (M-D) 2.8 (M) (M-D) RXR 4 (M-T) (T) 4.6/2.8 (M) (M-D) RXR 9C 4 (M-T) (T) 4.7/2.9 (M) (D) TR T 3 -GRIP1 4 (C) 4.8 (C) (C) TR T 3 -RXR 3.6 (D) 3.6 (D) nd TR T 3 -RXR-GRIP1 4.3 (C) 4.6 (C) (C) TR T 3 -RXR 9C-GRIP1 4.9 (C) 4.8 (C) nd Abbreviations: C, complex; D, dimer; M, monomer; nd, nondetermined; T, tetramer. a Data from GNOM (43). RXR-GRIP1 to form RXR homodimers (60, 61). The same low monodispersity was observed for TR T 3 - RXR-GRIP1 complex samples with and without 9C, also suggesting that the complexes represent stable forms for these proteins (Supplemental Table 2). To gather more structural parameters of NRs and NRcoactivator complexes, as well as data on the shape of complexes, we submitted samples with low polydispersity in DLS experiment to SAXS analysis. SAXS experiments confirmed essentially the same size parameters (R h )as found in DLS and SEC experiments (similar R h values, Table 2). Additionally, scattering curves and distance distribution functions [p(r)] from SAXS data show some interesting differences in complex sizes. As shown in Supplemental Figure 5 and Table 2, the heterodimer complex (TR 1-LBD T3-RXR -LBD-GRIP1) is little bigger than the homodimeric TR complex (Rg heterodimer GRIP nm, Rg homodimer GRIP nm). Also, our results show that the heterodimer is more globular, whereas the TR homodimer complex presents a more elongated shape (Supplemental Figures 5, A and B), confirming that GRIP1 is partially unfolded before association with TR (Krakty plot, Supplemental Figure 5C), as shown by our CD data. Therefore, the results depicted in Table 2 and Supplemental Figure 5 (Supplemental Material) indicate that 9C does not induce changes in the size of TR-RXR complexes, as previously reported for more truncated heterodimer constructions (33, 31). Stoichiometry of complexes To evaluate molecular masses and to confirm stoichiometry of complexes, as suggested by SEC, we used analytical ultracentrifugation (AUC). In sedimentation velocity experiments, it is possible to observe that TR presents two peaks, one for monomers and one for dimers [c(s) S and S (where S is the Svedberg unit for sedimentation coefficient), with measured masses of 38 9 kda and kda, respectively], corroborating DLS and SEC data (Table 2). RXR also presents itself as a mix of monomers and tetramers in solution [two peaks, c(s) S and S, respectively]. The TR-RXR presents itself mostly as heterodimers [c(s) S) and GRIP1 presented c(s) of S (Table 3)]. The complexes, TR T 3 -GRIP1 and TR T 3 -RXR 9C- GRIP1, also presented one peak with similar sedimentation coefficient, indicating similar sizes [c(s) S and S, respectively, Table 3]. The molecular masses calculated for complexes of homodimers and heterodimers plus GRIP1 was kda (TR 1- LBD T 3 -GRIP1) and kda (TR 1-LBD T 3 - RXR 9C-GRIP1), corresponding to a 2:1 stoichiometry or, alternatively, one TR homodimer or TR-RXR heterodimer bound to each GRIP1 (Figure 3). These data also reinforce the idea that coactivators containing more than one nuclear RID are sufficient to bind the homo- or heterodimers of NRs. Coactivator binding affinities To establish whether there are preferences in the binding affinity of TR homo- and TR-RXR heterodimers for GRIP1, we used fluorescence anisotropy assays. We also performed fluorescence anisotropy experiments using TR Table 3. Protein/Complex Analytical Ultracentrifugation Data C(s) (S) Molecular Mass, kda Oligomeric State TR Monomer Dimer TR-RXR Dimer RXR Monomer Tetramer Grip Dimer TR T 3 -Grip Complex TR T 3 -RXR 9C-Grip Complex Sedimentation coefficients and molecular weights as evaluated by sedimentation velocity experiments.

8 doi: /me mend.endojournals.org 265 Figure 3. Sedimentation coefficients as evaluated by AUC experiments. Sedimentation velocity analysis for proteins and complexes of truncated forms (short LBD) were performed in Sedfit program using the Lamm equation to fit the data (41). The sedimentation distribution plots of the TR 1 T 3 -GRIP1 complex show a narrow peak corresponding to one sedimentation specie (green peak, molecular mass kda), which indicates a 2:1 stoichiometry or, alternatively, one TR homodimer (two TR molecules) for each GRIP1. The same tendency was observed for TR T 3 -RXR 9C- GRIP1 complex (brown peak, molecular mass kda), indicating one TR-RXR heterodimer molecule to each GRIP1. (DBD-LBD) labeled with FITC as controls to show GRIP1 recruitment is T 3 dependent (Supplemental Figure 6 and Supplemental Table 3). We show that the TR-RXR heterodimer binds GRIP1 with almost the same affinity of the TR homodimers, which is a little lower for TR T 3 - RXR-GRIP1 binding (Figure 4A and Table 4). In other words, TR homo- and heterodimers bind GRIP1 with similar preferences. Moreover, we show that less truncated forms of homo- and heterodimers (containing DBD and LBD sequences) presented higher affinities to GRIP1 in comparison with truncated NRs (short LBD sequences), which, again, suggests additional contacts, or interactions between DBD/hinge and coactivator, also evidenced by our CD and DSF data (Table 1). 9C changes neither coactivator nor DNA recruitment We evaluated whether 9C modifies the behavior of heterodimers with regard to coactivator recruitment. To do that, we performed the same binding affinity assays of TR homo- and TR-RXR heterodimers for GRIP1 but in the presence of 9C. Our results show that 9C displays similar affinity for TR T 3 -RXR DBD-LBD, with or without GRIP1, suggesting that 9C is not important for coactivator recruitment. As we show in Table 4, truncated NR forms containing only the LBD presented lower affinity for GRIP1 after 9C addition, reflecting the DBD importance for complex stabilization. Figure 4. A, Fluorescence anisotropy curves of GRIP1 binding to TR T 3,TR T 3 -RXR, and TR T 3 -RXR 9C (DBD-LBD). Homo- and heterodimers bind to GRIP1 with the same higher affinity (Table 3). Also, 9C did not affects K d values, which indicates that its presence is not crucial for the binding of TR-RXR heterodimers to the coactivator. B, Fluorescence anisotropy curves of the TR T 3 -RXR-GRIP1 complex binding to DR4 in the presence or absence of 9C. The DR4 DNA sequence presents the same binding profile in the presence and absence of an RXR agonist (Table 4). Inserts, Same fluorescence plots, with logarithm scale in the x-axis. Moreover, because 9C does not change complex stability, size, and coactivator recruitment, we also investigated whether the RXR agonist modifies DNA recruitment by heterodimers. From our results on binding affinities of complexes to DR4 DNA in Figure 4B, we conclude that 9C also does not change DNA binding for TR 1 T 3 -RXR -GRIP1 complexes (Table 5). Cell assays suggest a hypothesis for the role of 9C in TR regulation To provide physiological data that correlate our biophysical results with the behavior of TR homo- and heterodimer complexes in cells, we performed transactivation assays in the context of HEK293 cell cultures. Previously our biophysical analysis show that TR homo- or heterodimer bind GRIP1, forming stable complexes with similar affinities. We observe that 9C binds to TR-RXR heterodimers and that its addition does not change size, shape, stability, or affinity to coactivator or to DNA binding. In our transfection results, TR activation is gradually increased in the presence of T 3, T 3 RXR, and T 3 RXR 9C, pointing out that 9C makes TR activation easier (Figure 5A). Also, we transfected RXR in the presence of T 3 and TR with 9C (Supplemental Figure 7) as negative controls, showing that 9C has no effect on TR and T 3 does not activate RXR. Although it was already

9 266 Fattori et al RXR Agonist Modulates Derepression of TR Mol Endocrinol, February 2015, 29(2): Table 4. Measured K d of TR T3 and TR T3-RXR Binding to GRIP1 in the Presence and Absence of 9C DL LBD K d, M n (Hill) K d, M n (Hill) TR T TR T 3 -RXR TR T 3 -RXR -9C demonstrated by our anisotropy fluorescence assays that 9C has no influence on the recruitment of coactivators by TR-RXR and because cellular assays showed that 9C increases heterodimer activation, we formulated a new hypothesis that 9C might influence corepressor displacement. To further check this hypothesis, we used TR mutants, which were expected to better interact with corepressor and coactivator, to decrease or to increase activation. One of these mutants, TR F451X, which lacks h12, induces repression through corepressor binding and does not recruit coactivators (51, 52). On the other hand, the TR V458M mutant facilitates coactivator binding by having one substitution in residues of the charge clamp, leading to derepression followed by higher activation in comparison with the TR wild type (53 55). Our results in Figure 5B show that TR F451X is repressed and neither T 3 nor RXR nor RXR T 3 was sufficient to activate the receptor because the absence of H12 strongly keeps TR-corepressor binding. Surprisingly, we observe a decrease in the basal repression of TR F451X- RXR after the addition of T 3 9C, suggesting corepressor dissociation. In parallel, the other mutant, TR V458M, was active in the presence of T 3 and increased its activation after RXR, T 3, and 9C addition, indicating that coactivator recruitment was more efficient when 9C T 3 were added to cell cultures (Figure 5C). Also, interestingly, the addition of 9C alone decreases basal repression of TR V458M, which constitutes further evidence of corepressor dissociation by this ligand. Coactivator and corepressor recruitment Finally, to substantiate the hypothesis suggested by our cell assays, namely that 9C helps in TR-RXR activation through corepressor dissociation without directly affecting coactivator binding, we performed fluorescence anisotropy assays with TR-RXR binding to corepressor (N-Cor) and with TR T 3 -RXR binding to coactivator Table 5. Measured K d of TR T 3 -RXR-GRIP1 binding to DR4 DNA in the Presence and Absence of 9C K d, M n Without 9C With 9C (SRC-1) peptides. For both peptides, the measured Kd was approximately 3.5 M, indicating similar binding affinities to the heterodimer (Figure 6A). Next, to observe the 9C effects under TR-RXR and TR T 3 -RXR binding to N-Cor and SRC-1 peptides, we titrated 9C in both complexes (TR-RXR N-Cor and TR T 3 -RXR SRC-1). In this case, we started to titrate 9C under the same samples used in the binding curves, which contained 10 M of heterodimer T 3 SRC-1 or 10 M of heterodimer N-Cor (Figure 6, B and C). We observed a decrease in anisotropy values when we added 9C to TR-RXR-N-Cor (in the presence or absence of T 3 ), suggesting that 9C binding is releasing the corepressor from the heterodimers (Figure 6B). Interestingly, the coactivator binding (TR-RXR-SRC-1, with or without T 3 ) was not affected by 9C, suggesting that the main recruitment of this specific coactivator identification is made by TR (Figure 6C). Finally, this fluorescence assay reinforces the idea that 9C helps into corepressor dissociation, whereas it did not change coactivator recruitment. Discussion TR-RXR heterodimer function has been intensively studied, and yet there are important gaps in our knowledge on the regulation of its mechanisms of action. Although RXR was first described in TR-RXR heterodimers, as a silent partner (3, 25, 27, 28, 30, 62 69), parallel studies in the literature showed the RXR agonist can alter TR- RXR heterodimer target gene transcription in response to this 9C s binding (34, 36, 70 72). In the first scheme, the proposed role for RXR was to facilitate TR binding to its cognate hormone responsive element, through heterodimerization (27, 30, 63, 69). Some studies have reported that RXR may indeed bind 9C in the context of TR-RXR heterodimers; however, 9C is not thought to activate retinoid signaling directly. Instead, it is considered to modulate TR repression, or activation, an action that is related to the phantom ligand effect (27, 31, 67). Concerns about the functions of 9C signaling via RXR mount in the literature, and as a consequence, its actual roles remain obscure in vertebrates (73). Furthermore, the multiple possible heterodimerization partners of RXR

10 doi: /me mend.endojournals.org 267 Figure 5. Transactivation assay of TR (wild type and mutants) and TR-RXR transfected in HEK293T cells in the presence and absence of ligands T 3 and 9C, using DR4 as a reporter. All the activation values presented were obtained by normalization with pbluescript control; hence, signals from endogenous NRs in DR4 were discounted. A, Transactivation assay of TR wild type showing T 3, RXR, T 3 RXR, and T 3 RXR 9C increased its activation. The heterodimer presented higher activation in the presence of both agonists (T 3 and 9C). *, P.05. B, Transactivation assay of TR F451X mutant. This TR mutant is repressed even in the presence of T 3 because of the lack of H12 that is important to coactivator binding surface. T 3, RXR and T 3 RXR did not affect TR F451X repression. However, 9C moderately increases TR F451X T 3 -RXR activation, indicating corepressor dissociation because coactivator binding is not privileged in this TR mutant. *, P.05. C, Transactivation assay of TR V458M, which make easier coactivator recruitment. T 3, RXR T 3, and RXR T 3 9C increase activation of this TR mutant, pointing to a faster coactivator recruitment, which is consistent with corepressor dissociation. *, P.05. These plots are average of at least six independent experiments. beg the question of whether there are any consistent mechanisms associated with RXR in the context of its various heterodimers (63, 69). The function of TR is a case in point and remains an open subject. Contradictory information has been reported, arising from various cell models. Although TR homodimers regulate promoters negatively, as repressors of gene transcription, TR-RXR heterodimers act mostly as activators bound to positive thyroid receptor response element, representing, perhaps, the most prevalent mode of regulation by thyroid receptors (4 9, 28). Here we describe how complexes are formed between TR, RXR, TR- RXR, and the GRIP1 coactivator and how 9C may play relevant roles in TR regulation via RXR binding. TR complex formation and stabilization Our first objective in this study was to characterize in detail complexes formed by bacterially expressed TR and RXR, both synthesized from cdna constructs that include far more amino acids than the widely used, but short, LBD versions. Using bacterial expression vectors for DBD- LBD TR, DBD-LBD RXR, and GRIP1 coactivator containing the three NR box motifs (RIDs, LXXLL motifs), we investigated the in vitro behavior of TR homo- and heterodimer complexes with GRIP1. Initially our CD analysis showed that TR, TR-RXR, and TR-RXR- GRIP1 display similar secondary structure, despite the fact that GRIP1 is basically unfolded in the absence of other proteins. Ultimately, this indicates that GRIP1 gains well-defined structural features when in complex with TR or TR-RXR (Figure 1), a conclusion further supported by our SAXS analysis (Supplemental Figure 5C and Supplemental Material). Our thermal unfold experiments revealed that less truncated NR forms (DBD-LBD) are more stable than the truncated NR form (LBD) (Table 1), showing that the DBD may contribute to LDB stability, probably due to induced structural rearrangements in protein structure. Therefore, these data provide objective evidence for additional relevant contacts between DBDs and coactivators because less truncated forms of NR present more stable complexes. Using SEC, DLS, and SAXS, we collected additional information about the size and shape of isolated receptors and coregulators as well as TR homo- and heterodimers in complex with GRIP1. The evaluated hydrodynamic radius for the complexes allowed us to propose that TR- GRIP1 and TR-RXR-GRIP1 are composed by one TR

11 268 Fattori et al RXR Agonist Modulates Derepression of TR Mol Endocrinol, February 2015, 29(2): Figure 6. Fluorescence anisotropy assays of TR-RXR heterodimer (LBD constructions) binding to coregulator peptides. A, Titration of the TR-RXR (LBD) in corepressor-labeled peptide (N-Cor) and in labeled coactivator peptide (SRC-1). All curves are an average of independent triplicates. Curves were fitted using the Hill equation, showing similar K d s for coactivator and corepressor peptide binding (K d M for TR-RXR N-Cor, and K d M for TR T 3 -RXR SRC-1). Insert, Same fluorescence plot, with logarithm scale in the x-axis. B, After TR-RXR binding to corepressor, 9C was titrated in these complexes containing 10 M of heterodimer N-Cor. Anisotropy decay at approximately 30% is observed after 9C addition, suggesting that 9C binding is releasing corepressor from our heterodimers. C, After TR T 3 -RXR binding to coactivator, 9C was titrated into the 10 M of heterodimer T 3 SRC-1 sample. No variation of anisotropy after 9C addition was observed, indicating that 9C does not influence recruitment of this coactivator (anisotropy value variations are within measurement errors). dimer (homo- or heterodimer) bound to one GRIP1 molecule (Table 2, Supplemental Figures 4 and 5). We further investigated this proportion by AUC experiments, and our results (Figure 3) show that TR-GRIP1 binding is asymmetric, with a stoichiometry of a dimer bound to one GRIP1 molecule, as was previously shown for peroxisomal proliferator-activated receptor (74), suggesting the possibility that one TR homodimer might be able to interact with more than one RID, but this issue needs to be further investigated. These data reinforce the notion that more complete coactivator sequences are sufficient to bind one NR homoor heterodimer and that this binding may be specific, depending on the NR, in contrast to what was reported for estrogen receptor-coactivator complexes (75 77). Finally, in the NR and NR-coactivator complex characterization assays, we derived binding affinities for AB TR homodimers or AB TR-RXR heterodimers to GRIP1, and the results indicate that the constants are of similar magnitude. These data indicate that GRIP1 does not display a binding preference for TR homo- or heterodimers (Figure 4A and Table 4), suggesting that the major interaction interface between TR homodimer, or TR heterodimer, and coactivators may be similar. NR complexes formed with minimal domains are less stable Even though the LBD is expected to display a relatively stable domain, due to its globularity (78), our analyses

12 doi: /me mend.endojournals.org 269 indicate that the stability of NR and coactivator complexes can be further increased when additional NR and coactivator domains are included. More specifically, our thermal unfolding analyses showed that complexes formed by truncated, minimal domains are less stable than those that include DBD plus LBD. Also, our affinity data from TR LBD and TR-RXR LBD binding to GRIP1 (Table 4 and Figure 4A) agrees with this hypothesis, showing higher affinity for more complete NR constructions over those containing only LBD. This suggests that the presence of DBD and hinge domains increases TR stability after GRIP1 binding, also enhancing the affinity for complex formation (Table 4). The presence of intra- and interdomain communication and allosteric modifications have already been described for NRs, pointing out that ligand binding may promote structural changes in LBD, which modify the coactivator binding interface (79). Likewise, direct interactions between DBD and LBD have also been described as important to specify DNA binding (18, 33). Here we show that apart from the LBD, the DBD and hinge also play considerable roles in the stabilization of more complete NR-coactivator complexes. It remains to be determined whether this effect is dependent on direct binding, but it seems likely that the presence of DBD/hinge allows TR dimers to achieve more appropriate conformations, which are reflected in a better interaction with GRIP1. The effects of 9C in TR-RXR-GRIP1 complex conformation One of the main goals of this study was to investigate whether the TR-RXR heterodimer is permissive to 9C. Our fluorescence quenching assays show, for the first time, that 9C can physically bind TR-RXR heterodimers (Figure 2 and Supplemental 3). Also, we observed that the specific conformational changes induced by 9C and measured around RXR W355 are maximum when in the context of the TR-RXR-GRIP1 complex. After these results, we conclude that 9C causes local conformational changes in heterodimer structure, which were not observed in less sensitive assays such as gel filtration, SAXS, and DLS. As shown in our biophysical assays, we first compared whether 9C provokes any changes in the global structural parameters of TR-RXR and TR-RXR-GRIP1 complexes. Our biophysical data show that 9C did not change any global structural parameter of AB TR-RXR heterodimers, even in the presence of GRIP1 (Table 2). Moreover, the measured binding affinities of TR-RXR to GRIP1 and of TR-RXR-GRIP1 to DNA were very similar after 9C addition (Tables 4 and 5). All these results show that the RXR agonist did not cause sufficient changes in the TR-RXR structure, which keep the size, stability, and shape of the complex. For these reasons we conclude that 9C is not sufficient to modify physical affinities for complex formation or for DNA binding. These findings contrast with some studies that suggest that 9C disrupts the heterodimer (31) or that it changes affinities for coactivator recruitment (33, 31, 34). However, it is important to consider that different protein architectures and the use of minimal domains, as opposed to full-length constructions, could lead to different results. Additionally, differences in cell context might explain these differences, as previously described (34). 9C does not disrupt DBD-LBD heterodimers Our fluorescence data from TR and TR-RXR binding to GRIP1 showed differences in the affinities of these NRs when we compared LBD and DBD-LBD constructions (Table 4). We observed that the TR-RXR LBD forms a complex with GRIP but with smaller affinity. Also, our results indicate that 9C interferes in the cooperativity of the TR-RXR-GRIP1 LBD complex, suggesting an anticooperative behavior, which was not observed for DBD- LBD constructions. The anticooperativity and lower affinity should be explained by structural instabilities caused by the 9C addition to TR-RXR LBD. Interestingly, the crystallographic structure of TR T 3 -RXR 9C revealed that there are minimal changes in the overall LBD conformation after heterodimerization and that 9C disrupts the structural stability of TR (31). Altogether, it is possible to propose that 9C may play a role in heterodimer dissociation and that the TR-RXR heterodimer is not permissive to 9C. Here we explain these dissociation effects, revealing that the instability of LBD heterodimer is a result of the lack of DBD, which seems to be important to keep other interaction interfaces among NRs and coactivator, as reported for other NR-corepressor complexes (80 82). Our results showed that the addition of DBD/hinge in protein composition abolished this anticooperative behavior and constitute further evidence of the importance DBD/hinge in keeping the stability of NR-coactivator complexes (Table 3). Naturally all these results indicate the necessity of additional efforts in structural studies of full-length nuclear receptors and more inclusive, less truncated coregulator constructions, which will provide detailed information about quaternary structure and NR complex formation with DNA and coregulators. 9C is important to corepressor dissociation The results from our cellular assays are consistent with our binding affinity data. Collectively they indicate that 9C binding to RXR in the context of TR-RXR heterodimers plays no role in heterodimer affinity to target