Structure-Function Analysis of the Rev-erbA and RVR Ligand-Binding Domains Reveals a Large Hydrophobic Surface That Mediates Corepressor Binding and a Ligand Cavity Occupied by Side Chains

Jean-Paul Renaud, Jonathan M. Harris, Michael Downes, Les J. Burke and George E .O. Muscat

Centre Nationale de la Recherche Scientifique UPR9004 Laboratoire de Biologie et Genomic Structurales (J.P.R.) Institut de Génétique et Biologie Moléculaire et Cellulaire F-67404 Illkirch, France
University of Queensland (J.M.H., L.J.B., M.D., G.E.O.M.) Institute for Molecular Bioscience (I.M.B.) Centre for Molecular and Cellular Biology Ritchie Research Laboratories B402A St. Lucia, 4072 Queensland, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3–4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13{Delta}1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Members of the nuclear receptor (NR) superfamily bind specific DNA elements and can function as ligand-regulated transcription factors (1, 2). This group includes the orphan receptors, which have no known ligands in the classical sense and appear to be the ancient progenitors of this receptor superfamily (3). The Rev-erb family of proteins, Rev-erbA/ear-1 (4, 5, 6, 7) and RVR/Rev-erbB/BD73 (8, 9, 10), are orphan members of this superfamily. These two proteins are highly conserved in the DNA-binding domain (DBD) (95%) and the ligand-binding domain (LBD) (70%); however, major differences between the two isoforms occur within the hypervariable A/B and D regions of the proteins.

Rev-erbA and RVR bind as monomers to the nuclear receptor half-site motif, RGGTCA flanked 5' by an AT-rich sequence [(A/T)6RGGTCA], and as dimers to a novel direct repeat motif separated by 2 bp [Rev-DR-2, (A/T)4 AGGTCA CT AGGTCA] (11, 12). The Rev-erb family members function as ligand-independent dominant transcriptional repressors (13, 14, 15) and the LBDs of Rev-erbA and RVR encode active transcriptional silencers (14, 15). Furthermore, we demonstrated that efficient repression (of GAL4VP16-mediated transactivation) is independently mediated by approximately 35 amino acids (aa) between aa 455–488 in the LBD of Rev-erbA and aa 416-449 of RVR (14, 15). This repression domain contains the LBD-specific signature motif (F/W)AKXXXXFXXLXXXDQXXLL (16) and spans H3–H5.

Ligand-independent repression of transcription by Rev-erbA and RVR is mediated by the nuclear receptor corepressor N-CoR and its variants RIP13a and RIP13{Delta}1 (17, 18, 19, 20, 21, 22). Detailed analysis of the corepressors identified a C-terminal receptor interaction domain (RID) that consists of two interaction domains, ID-I and ID-II, that efficiently interact with the ligand-regulated and orphan nuclear receptors (20, 21, 22). Furthermore, it has been demonstrated that RVR and Rev-erbA interact very efficiently with the RID from N-CoR and RIP13a, although they preferentially interact with the RID from RIP13{Delta}1 (20). Moreover, it was demonstrated that the LBD of RVR and Rev-erbA was necessary for the interaction with corepressors (20).

The Rev-erbA LBD regions interacting with N-CoR have also been identified. Initially Lazar and colleagues delineated two regions in Rev-erbA by in vitro studies, domain X (aa 407–418) and domain Y (aa 602–614) (21), that were shown to be necessary for the efficient interaction with the corepressor, N-CoR. These regions correspond to the extra domain and H11, respectively (see Fig. 1Go). However, in a follow-up study by the same group, they suggested that the X domain was not required for corepressor binding in vivo (23). In a later study (24), we demonstrated that corepressor interaction region 2 (CIR-2)/Y-domain in H11 of Rev-erbA and RVR was necessary for the interaction with the corepressor, N-CoR, and the variant, RIP13{Delta}1. Furthermore, we also defined a novel domain (CIR-1), which corresponds to H3 in both Rev-erbA and RVR, that was necessary for the efficient interaction of both orphan nuclear receptors with both corepressors, N-CoR and RIP13{Delta}1. Moreover, we used mutagenesis to demonstrate that CIR-1/H3 and CIR-2/Y-domain/H11 in both receptors are necessary for 1) the interaction with the corepressors N-CoR and RIP13{Delta}1, and 2) transcriptional repression of a physiological target, the human (h) Rev-erbA promoter (24). F439 in Rev-erbA and F402 in RVR were critical residues in supporting corepressor interaction. This suggested that a minimal region containing CIR-1 and CIR-2, in H3 and H11, respectively, was necessary for corepressor binding and transcriptional repression. It was hypothesized that CIR-1 and CIR-2 are juxtaposed in the putative three-dimensional tertiary structure of these orphan receptors and probably form a single contact interface that interacts with the nuclear receptor corepressors.



View larger version (46K):
[in this window]
[in a new window]
 
Figure 1. Sequence Alignment between hRev-erbA{alpha} (REV), mRVR (RVR), and hRAR{gamma} (RAR)

Amino acid identity (stars) and similarity (dots) between hRev-erbA{alpha} and mRVR and between all three sequences are shown above and below the alignment, respectively. hRev-erbA{alpha} and hRAR{gamma} numberings appear above and below the alignment, respectively. Thin boxes indicate the secondary structure elements in the hRAR{gamma} crystal structure (29 ). The cavity-forming residues in hRAR{gamma} and the homologous residues in hRev-erbA{alpha} and mRVR are shaded. The CIR-1 and CIR-2 interaction domains with N-CoR (24 ) are underlined in the hRev-erbA{alpha} sequence (thick broken lines). Thick boxes indicate the residues from the hydrophobic surface in the hRev-erbA{alpha} and mRVR homology models that have been mutated in the present study.

 
However, further experimentation was required to verify this hypothesis. To resolve these contradictions in the structure and specificity of the orphan nuclear receptor-corepressor interaction, three dimensional analysis of Rev-erbA and RVR was required. The crystal structure of several NR LBDs has already been determined and reviewed recently (Ref. 25 and references therein). Furthermore, the structure and specificity of ligand-activated NR-coactivator interactions have been resolved. These studies suggested that a hydrophobic grove comprised of H3-H4 and H12 in ER{alpha}, TRß, and peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) mediates the interaction to the coactivators steroid receptor coactivator 1 (SRC-1) and SRC-2/glucocorticoid receptor interacting protein 1 (GRIP-1) (26, 27, 28). However, no structural information is available yet on the interface between ligand-activated or orphan NRs with corepressors. Here we describe homology models of the Rev-erbA and RVR LBDs built using as a starting model the crystal structure of the human retinoic acid receptor-{gamma} (hRAR{gamma}) LBD in complex with all-trans-retinoic acid (29). These models suggested that Rev-erbA/RVR may have no endogenous ligand. Moreover, the Rev-erbA/RVR LBD formed a very hydrophobic surface that mediated corepressor binding. We mapped this site by the construction of 20 site-specific mutations in Rev-erbA/RVR that spanned H3, loop 3–4, H4, and H11 and used these mutants in a variety of in vitro and in vivo two-hybrid interaction studies in mammalian cells to complement the modeling of the LBD structures.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Homology Modeling of the Rev-erbA and RVR LBDs
The sequence alignment of hRev-erbA{alpha}, murine (m)RVR, and hRAR{gamma} LBDs is presented in Fig. 1Go, in good agreement with the general alignment of all NR LBD sequences previously published (16). Residues 430–614/393–576/221–405 (hRev-erbA{alpha}/mRVR/hRAR{gamma} numbering) comprising the region between H3 and H11 of the LBD were aligned without problem as no insertion or deletion occurred in these regions. On the other hand, residues 281–301/243–263/182–202 encompassing H1 at the N terminus of the LBD were aligned thanks to the H1-specific motif AHXXT, present in many NR LBDs and responsible for the attachment of H1 to the LBD core (16). In Rev-erbA and RVR, this motif was found 120 aa upstream compared with RAR (in hRev-erbA{alpha}, an isoleucine replaces the conserved threonine). Interestingly, the extra domains in Rev-erbA and RVR (residues 302–429 and 264–392, respectively) have almost identical lengths (128 and 129 aa, respectively), but their sequences diverge widely, pointing to a differential role in the regulation of the activity of these receptors. In the aligned regions, the hRev-erbA{alpha}/mRVR LBDs show 17/18% sequence identity (30/32% sequence homology) relative to the hRAR{gamma} LBD and 70% sequence identity between each other (Fig. 1Go).

The template for homology modeling was the crystal structure of the hRAR{gamma} LBD (29). The extra domains were not included so the homology models comprised residues 281–301 and 430–614 for Rev-erbA, and residues 243–263 and 393–576 for RVR. After amino acid replacement according to the present alignment, an energy minimization was performed with X-PLOR (30) using a simulated annealing method for side chain building (31, 32) (see Materials and Methods for details). The final models were analyzed with PROCHECK (33), which shows that no residue is found in the disallowed regions of the Ramachadran plots and that the main chain and side chain parameter statistics are inside the range of, or better than, the statistics derived from crystal structures solved at a resolution of 2 Å. After different trials, it was found that the best superposition of Rev-erbA/RVR onto RAR (Fig. 2AGo) was obtained manually by optimally superposing the central H4–H5 helices only (residues 465–487/428–450/256–278, hRev-erbA{alpha}/mRVR/hRAR{gamma} numbering). In this way, H8 and H9 are found almost unchanged, in good agreement with the fact that H4-H5, H8, and H9 form together the static part of the LBD. The main change is due to the absence of H12, causing a tilt of H11 toward H3 and a concomitant inward shift of the H6-H7 region and of the ß-sheet, in the direction of H11. As a result, H11 of Rev-erbA/RVR lies in between H11 and H12 of RAR, making numerous side chain contacts with H3 (see below).



View larger version (56K):
[in this window]
[in a new window]
 
Figure 2. Homology Models of the Rev-erbA and RVR LBDs

A, Stereoview of the superposition of the C{alpha} traces of the hRev-erbA{alpha} (green) and mRVR (red) LBD homology models with the hRAR{gamma} crystal structure (blue). The long insertion between H1 and H3 has not been modeled. B, Stereoview of the superposition of the putative ligand pockets of hRev-erbA{alpha} (green backbone, blue side chains) and mRVR (yellow backbone, red side chains). There is no room for a ligand as the side chains of the residues lining the pocket occupy all the cavity. The residual cavity has a probe-occupied volume of 16 Å3 in hRev-erbA{alpha} and cannot be detected in RVR. The Rev-erbA residues are labeled. C, Stereoview of the superposition of the putative ligand pocket of hRev-erbA{alpha} (green backbone, blue side chains) and the ligand-binding site of hRAR{gamma} (pink backbone, purple side chains) with all-trans retinoic acid (yellow). The probe-occupied cavity of hRAR{gamma} (418 Å3) is shown in light blue. The side chains of RAR shown are those homologous to the 11 hydrophobic residues in Rev-erb/RVR that cluster in the putative ligand pocket (see Fig. 1Go for the alignment and Fig. 2BGo for the labeling, and see text for details). The orientation is the same as in Fig. 2BGo but the scale is slightly smaller. [This figure was prepared with SETOR (57 ).]

 
Rev-erbA and RVR May Lack Ligands
Rev-erbA/RVR lack H12 at the LBD C terminus, which has been shown to be essential for ligand-dependent activation of transcription (Ref. 25 and references therein). It is thought that ligand binding triggers a conformational change that repositions H12, resulting in the creation of a new surface to which coactivators can bind (16, 29). As noted previously by several authors, the absence of H12 and the fact that Rev-erbA/RVR act as constitutive repressors suggest that these receptors have no need of a ligand.

Indeed, in the Rev-erbA/RVR models, the ligand cavity is occupied by side chains, with no room left for a potential ligand (Fig. 2BGo). This results from both the presence of bigger side chains at positions homologous to the pocket-forming residues in RAR{gamma} and the shift of some secondary structure elements (Figs. 1Go and 2Go). Among these 21 positions, 9 residues are identical: F201/300/262, W227/436/399, F230/439/402, L271/480/443, M272/481/444, R274/483/446, F288/497/460, G303/512/475, and L307/516/479 (hRAR{gamma}/hRev-erbA{alpha}/mRVR numbering). Three differences do not significantly affect the size of the cavity: K236 is replaced by a proline in Rev-erbA/RVR (P445/408), R278 by a leucine (L487/450), and R396 by a lysine in Rev-erbA (K605) and a glutamate in RVR (E568), but these side chains do not point into the cavity. Two residues are smaller in Rev-erbA/RVR: C237 is an alanine (A446/409), and F304 is a methionine in Rev-erbA (M513) and an alanine in RVR (A476). On the other hand, seven residues are bigger in Rev-erbA/RVR: A234, L268, I275, and L400 are all replaced by phenylalanines (F443/406, F477/440, F484/447, and F609/572, respectively), G393 by a histidine (H602/565), and S289 and A397 by leucines (L498/461 and L606/569, respectively). All these seven, mostly aromatic side chains cluster to form a large hydrophobic core together with F439/402, with M513/A476 and L516/479 brought in the vicinity by the H6-H7 shift, and with F497/460 brought deeper into the pocket by the inward shift of the ß-sheet compared with hRAR{gamma} (Fig. 2Go, B and C). At the center, the volume of the retinoic acid cavity in hRAR{gamma} is filled up by the F484/447, L498/461, M513/A476, L516/479, and H602/565 side chains (Fig. 2CGo). Calculations with VOIDOO (34) indicate that the residual ligand-binding pocket has a probe-occupied volume of 16 Å3 in hRev-erbA{alpha} (compared with 418 Å3 in hRAR{gamma}), which is not enough to accommodate a ligand. In mRVR, no residual cavity at all was found by VOIDOO. This seems paradoxical because Rev-erbA and RVR differ at only two positions over 21 at the level of the pocket (Fig. 1Go): first, K605 in Rev-erbA corresponds to E568 in RVR, but these side chains do not point into the cavity (see above); second, M513 in Rev-erbA corresponds to A576 in RVR, and this time the side chains do point into the cavity, so the RVR residual pocket should be larger. But in fact, the methionine-to-alanine change is compensated by slight shifts of the neighboring F402, L471, and L480 side chains. In addition, a shift of G512 in RVR relative to the homologous G475 in Rev-erbA allows the flipping of F572 aromatic ring into the pocket compared with the homologous F609 in Rev-erbA, also contributing to compensate the methionine-to-alanine change (Fig. 2BGo).

In summary, the present homology models strongly suggest that Rev-erbA and RVR lack a ligand, in line with the absence of the activation helix H12. However, we cannot preclude the existence of a novel regulatory molecule.

The Rev-erbA and RVR LBDs Possess a Highly Hydrophobic Surface Comprising H3, Loop 3–4, H4, and H11
A striking feature in the model is a large hydrophobic surface comprising the side chains of the following residues: W436/399, F443/406, V447/410, and V451/414 in H3, R461/424 in loop 3–4, V469/432 and K473/436 in H4, and L606/569, F609/572, and R610/573 in H11 (hRev-erbA{alpha}/mRVR numbering) (Fig. 3Go). Comparison of the Rev-erbA/RVR and RAR C{alpha} traces (Fig. 2AGo) shows that the absence of H12 explains not only the surface exposure of these residues, but also the shift of several secondary structure elements. In Rev-erbA, the tilt of H11 brings it in close contact with H3: H602 makes van der Waals interactions with F439 and F443; L606 with F443; F609 with W436, F439, and F443; and R610 with W436 (Figs. 2BGo and 3BGo). By contrast, H3 and H11 in hRAR{gamma} are more distant, making only one van der Waals contact (between W227 and L400). This is due not only to the shift of H11, but also to side chain differences between homologous residues: F443, L606, and F609 in Rev-erbA correspond to A234, A397, and L400 in hRAR{gamma}, respectively. Thus, it seems that H3 and H11 in Rev-erbA form together a continuous hydrophobic surface, extended by side chains from loop 3–4 and H4. In RVR, the same van der Waals contacts as in Rev-erbA are observed, except between F406 and H565 and between F406 and F472 because of a flip of F406 aromatic ring, and between W399 and K573 due to a different side chain orientation of K573 compared with Rev-erbA R610 (Figs. 2BGo and 3BGo). Nevertheless, H3 and H11 still form together a hydrophobic surface, very similar to that in Rev-erbA.



View larger version (76K):
[in this window]
[in a new window]
 
Figure 3. A Hydrophobic Surface in the Rev-erbA and RVR LBD Homology Models, Including Residues from H3, Loop 3–4, H4, and H11, Is the putative N-CoR Interaction Surface

A, Electrostatic potential surfaces of the Rev-erbA and RVR LBDs, calculated using GRASP (56 ). The regions of negative potential are shown in red and the regions of positive potential are shown in blue. The hydrophobic surfaces appear as the large white regions. The positively charged residues contributing to this surface are labeled. In the models, their charged moiety interacts for all of them with polar, uncharged groups through hydrogen bonds; therefore their charge is not neutralized locally and gives rise to a strong, positive potential region (deep blue patches), except for R610 in Rev-erb which makes a salt bridge with the C-terminal Q614 carboxylate and hence appears as a faint blue patch (see text). B, Superposition of the hydrophobic surfaces in the Rev-erbA and RVR LBDs showing the residues that have been mutated to alanine in the present study. Only H3, loop 3–4, H4, and H11 are shown for clarity. [Panel B was prepared with SETOR (57 ).]

 
In the models, the hydrophobic part of the side chain of the three positively charged residues (R461, K473, and R610) is exposed, and their polar extremity is stabilized by polar interactions with other surface residues, but the conformation of charged surface residues is probably not very reliable since the minimization is carried out in the absence of solvent and ions. Furthermore, they may as well be involved in polar interactions with N-CoR residues within the complex. For instance, the conserved lysine and the conserved glutamine in the LBD signature are hydrogen bonded in the RAR LBD structure (29), but the lysine is engaged in the binding of the LXXLL-containing helix in the complex with the coactivator as shown by mutation studies (35) and structural studies (26, 27, 28). In any event, all the above mentioned residues have been mutated to alanine, and the mutant LBDs have been assayed for ability to interact with the corepressors N-CoR and RIP13{Delta}1 with in vitro glutathione S-transferase (GST)-pulldown experiments and in vivo using mammalian two-hybrid assays.

In Vitro Mutational Analysis of the Hydrophobic Surface in the Rev-erbA LBD: H3, Loop 3–4, H4, and H11 Are Directly Involved in Corepressor Binding
We have presented a homology model of the Rev-erbA and RVR LBDs, showing that Rev-erbA and RVR most probably lack a ligand, in good agreement with their dominant silencing function. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3–4, H4, and H11. We used neutral alanine substitution mutagenesis to investigate the involvement of key residues (as described above), in the physical association with corepressor, N-CoR, and its variant, RIP13{Delta}1. We measured and examined the interaction between Rev-erbA and RVR, and the RIDs from N-CoR and RIP13{Delta}1, by in vitro GST pulldown assays and mammalian two-hybrid in vivo interaction assays, as a tool to confirm and extend the structural predictions. We made 20 mutants, 10 in Rev-erbA and 10 in RVR at homologous positions (see Figs. 1Go and 3BGo). The mutations in hRev-erbA{alpha} were W436A, F443A, V447A, V451A, R461A, V469A, K473A, L606A, F609A, and R610A. The mutations in mRVR were W339A, F406A, V410A, V414A, R424A, V432A, K436A, L569A, F572A, and K573A.

We had previously demonstrated that full-length Rev-erbA and RVR efficiently interacted with the receptor interaction domains (RIDs) from N-CoR and RIP13{Delta}1. The RIDs from N-CoR and RIP13{Delta}1 are similar. However, the RID from RIP13{Delta}1 has an internal deletion of 120 amino acids (18) in ID II. Furthermore, we had demonstrated that both H3 and H11 in these orphan NRs was necessary for the interaction with the N-CoR and the RIP13{Delta}1 RIDs (20). Hence, we embarked on further direct in vitro binding GST-pulldown assays and mammalian two-hybrid analysis to identify the amino acid residues on the hydrophobic surface rigorously and define the specific helices that mediated corepressor binding. Furthermore, we used the GST pulldown assay to determine which receptor residues were responsible for discrimination between N-CoR and RIP13{Delta}1 RIDs.

We then used a biochemical approach, the in vitro GST pulldown assay, to investigate which residues in the hydrophobic surface impacted on the ability of the orphan receptor to specifically bind to the N-CoR and RIP13{Delta}1 RIDs.

Glutathione agarose-immobilized GST-N-CoR and RIP13{Delta}1 RIDs were tested for direct interaction with in vitro 35S-radiolabeled native and mutant Rev-erbA proteins (Fig. 4Go). The GST-N-CoR/RIP13a and RIP13{Delta}1 RIDs showed a direct interaction with native Rev-erbA. We observed that mutations F443A, V447A, and V451A in H3 had a significant effect on the ability of Rev-erbA to physically interact with the N-CoR RID [our previous study had demonstrated that a double mutation F339A/S440A in H3 had the same effect (24)]. R461A and V469A in loop 3–4 and H4, respectively, also affected binding to N-CoR/RIP13a RID. The point mutations in H11 did not affect corepressor RID binding; however, mutations L606A and F609A reduced the specificity of the interaction with the N-CoR/RIP13a RID, i.e. increased binding to GST alone.



View larger version (91K):
[in this window]
[in a new window]
 
Figure 4. Characterization of the Impact of Mutations in RVR and Rev-erbA on the Interaction with N-CoR and the Variant RIP13{Delta}1 RIDs in Vitro

A, Sequence alignment of the RVR and Rev-erbA LBDs with the helices boxed; the residues mutated in each receptor are denoted by an asterisk. B, In vitro biochemical interaction of the ReverbA and RVR with the corepressors N-CoR and RIP13{Delta}1. Wild-type Rev-erbA and RVR and the corresponding site-specific single mutations that spanned H3-H4 and H11 were radiolabeled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone, GST-N-CoR, and GST-RIP13{Delta}1. Inputs of the radiolabeled 35S-methionine protein are also shown and contain approximately 10% input.

 
In contrast, only V447A in H3, R461A ,and V469 in loop 3–4 and H4, respectively, had effects on RIP13{Delta}1 RID binding, relative to wild-type Rev-erbA binding. Interestingly, the in vitro interaction of Rev-erbA to the RIP13{Delta}1 RID relative to the N-CoR RID is more robust and refractory to the effect of some mutations (e.g. F443A and V451A in H3) that significantly impair binding to the N-CoR RID (Fig. 4Go).

In Vitro Mutational Analysis of the Hydrophobic Surface in the RVR LBD: H3, Loop 3–4, H4, and H11 Are Directly Involved in Corepressor Binding
We then investigated the potential of various single-point mutations in H3, loop 3–4, H4, and H11 of RVR [in the context of the full-length receptor] to interact with the N-CoR and RIP13{Delta}1 RIDs in the GST-pulldown assay (Fig. 4Go). Interestingly, V410A and V414A in H3 of RVR had minor and significant effects, respectively, on the interaction with the N-CoR and RIP13{Delta}1 RIDs (our previous study demonstrated that the single-point mutation, F402P, but not E400K, in H3 had a dramatic and significant effect on the ability of RVR to physically interact with both the N-CoR and RIP13{Delta}1 RIDs).

Curiously, R424A in loop 3–4 significantly increased binding to the N-CoR RID, but not to the RIP13{Delta}1 RID. Mutations V432A and K436A in H4 did not effect binding to the N-CoR RID. However, they significantly affected binding to the RIP13{Delta}1 RID (relative to the native RVR). The mutations (L569A, F572A, and K573A) in H11 of RVR did not affect binding to the N-CoR RID (although K573A had a weak effect). However, these mutations had a minor to significant effect on the ability of RVR to bind to the RIP13{Delta}1 RID.

In summary, these in vitro biochemical results suggest that H3, loop 3–4, H4, and H11 are part of a hydrophobic surface that mediates binding to the corepressors. The in vitro data support the hypothesis that there are differences between the interaction of Rev-erbA and RVR with the corepressors. Furthermore, the assay suggests that the orphan NR hydrophobic surface (H3, loop 3–4, H4, and H11) encodes the potential to interact differentially with the different corepressor isoforms.

In Vivo Mutational Analysis of the Hydrophobic Surface in the Rev-erbA LBD: H3, Loop 3–4, H4, and H11 Are Directly Involved in Corepressor Binding
Many studies have observed that in vitro biochemical data do not always correlate with the in vivo analysis; hence we embarked on an in vivo interaction analysis. We had previously demonstrated that full-length Rev-erbA efficiently interacted with the N-CoR and RIP13{Delta}1 RIDs, which are very similar, except for an internal deletion of 120 amino acids in RIP13{Delta}1 ID-II (18). Furthermore, we have previously shown that H3 and H11 in this orphan receptor were necessary for the interaction with the N-CoR and RIP13{Delta}1 RIDs (20). Hence, we embarked on further mammalian two-hybrid analysis, to identify the amino acid residues rigorously on the hydrophobic surface that were critical to the physical association with the corepressors. The chimeric constructs consisting of the yeast GAL4 DBD fused to the N-CoR and RIP13{Delta}1 RIDs were each individually transfected/expressed in cells with a set of cotransfected chimeric constructs containing mutations of Rev-erbA linked to the transactivation domain of VP16 (Fig. 5AGo). We observed that mutations W436A (H3), V451A (H3), R461A (loop 3–4), V469A (H4), L606A (H11), and R610A(H11) all had a dramatic (almost a knockout) effect on the ability of Rev-erbA to interact with the N-CoR and RIP13{Delta}1 RIDs, respectively (our previous study demonstrated that the double mutant F339A/S440A in H3 similarly ablated the in vivo interaction between Rev-erbA and the corepressors). The mutations at F443A(H3) and V447A(H3) had a weak to significant effect on the ability of Rev-erbA to interact with the N-CoR and RIP13{Delta}1 RIDs. The changes at positions K473A and F609A had a minimal to insignificant effect on the ability of Rev-erbA to interact with either the N-CoR and RIP13{Delta}1 RIDs (Fig. 5AGo).



View larger version (57K):
[in this window]
[in a new window]
 
Figure 5. Characterization of the Impact of Various Site-Specific Mutations in the H3, Loop 3–4, H4, and H11 Regions of Rev-erbA and RVR on Corepressor Recruitment

A, JEG-3 cells were cotransfected with the indicated wild-type and mutant Rev-erbA receptors linked to the transactivation domain of VP16 (+) in the presence of either the GAL4-N-CoR or RIP13{Delta}1 RID and pG5E1bLUC reporter plasmid. B, Whole-cell extracts from cells transfected with the indicated wild-type and mutant Rev-erbA and RVR receptors linked to VP16 were analyzed on Western blots using a polyclonal antibody to the VP16 transactivation domain (Santa Cruz no. SC-1728). The positions of the expressed proteins are indicated. C, JEG-3 cells were cotransfected with the indicated wild-type and mutant RVR receptors linked to the transactivation domain of VP16 (+) in the presence of either the GAL4-N-CoR or RIP13{Delta}1 RID and pG5E1bLUC reporter plasmid. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to LUC activity obtained after transfection of the GAL4-corepressor RID and the VP16 vector alone arbitrarily set to 1.0.

 
Western analysis of the VP16 chimeric wild-type and mutant Rev-erbA proteins demonstrates that the mutants express at levels similar to wild-type protein. Moreover, the Western shows a lack of degradation and highlights the stability of the proteins (Fig. 5BGo).

These results suggest that the functional interaction, in vivo, is dependent on the hydrophobic surface of the Rev-erbA LBD that encompasses H3, loop 3–4, H4, and H11. The in vitro and in vivo analysis of the interaction between the corepressors and Rev-erbA strongly suggests that the hydrophobic surface of the Rev-erbA LBD that encompasses H3, loop 3–4, H4, and H11 mediates the transmission of transcriptional signals from the orphan NR to the corepressor.

In Vivo Mutational Analysis of the Hydrophobic Surface in the RVR LBD: H3, Loop 3–4, H4, and H11 Are Directly Involved in Corepressor Binding
We then investigated the potential of various single-point mutations in the H3, loop 3–4, H4, and H11 of RVR [in the context of the full-length receptor] to interact with the N-CoR and RIP13{Delta}1 RIDs in the mammalian two-hybrid assay. The chimeric construct consisting of the yeast GAL4 DBD fused to the N-CoR and RIP13{Delta}1 RIDs were individually expressed in cells with a set of chimeric constructs containing mutations of RVR linked to the transactivation domain of VP16. Interestingly, only the mutations V414A (H3), V432A (H4), and K436A (H4) had a significant impact and reduced the ability of RVR to interact with the N-CoR RID (Fig. 5CGo). However, the interaction of RVR with the RIP13{Delta}1 RID was less robust; we observed that mutation F406 in H3 dramatically reduced the corepressor interaction, and mutations V414A (H3), R424A (loop 3–4), V432A (H4), K436A (H4), and L569A (H11) significantly inhibited the interaction with the RIP13{Delta}1 RID. Our previous study demonstrated that the single-point mutation F402P in RVR ablated the interaction between RVR and both corepressors (N-CoR and RIP13{Delta}1) in the in vivo two-hybrid assay. The RVR mutants are expressed at a similar level to wild-type RVR as shown by Western blotting (Fig. 5BGo). Additionally, a lack of RVR degradation products indicates that the wild-type and mutant proteins have similar stabilities (Fig. 5BGo).

The analysis of the interaction between the corepressors and RVR suggests that the hydrophobic surface of the RVR LBD that encompasses H3, loop 3–4, H4, and H11 is directly involved in corepressor recruitment and binding.

Transcriptional Repression Studies of a Physiological Target, the Human Rev-erbA Promoter
Laudet and colleagues (58) previously characterized the hRev-erbA{alpha} gene promoter, a natural target of Rev-erb and RVR. We have used this physiological target of Rev-erb and RVR action as a tool to investigate the effect of point mutants in H3 and H11 on RVR-mediated silencing of promoter expression (24, 58). Hence, we used this assay as a tool to provide an insight into the impact of the mutations spanning H3, loop 3–4, H4, and H11 on the function of the orphan receptors as transcriptional silencers. Native Rev-erb and the various point mutations were investigated for their ability to repress the Rev-erbA{alpha} promoter linked to the luciferase reporter in C2C12 myogenic cells. Rev-erb and RVR are known to be expressed in mouse C2C12 muscle cells and to repress the ability of these cells to differentiate. As previously observed, native Rev-erbA{alpha} represses transcription of its own promoter by approximately 3-fold (24, 58). The mutations in hRev-erbA{alpha} at positions F443A and V447A ablated the ability of Rev-erb to silence promoter expression (Fig. 6AGo). V451A and F609A mutations did not ablate silencing but significantly reduced the ability of Rev-erbA to silence promoter expression. Mutation of W436A, R461A, V469A, and K473A reduced the ability of Rev-erbA to repress transcription by 2-fold. The L606A and R610A mutations did not significantly affect silencing of transcription.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 6. Repression of the hRev-erbA{alpha} Gene Promoter by hRev-erbA{alpha} and mRVR

C2C12 cells were cotransfected with the indicated wild-type and mutant Rev-erbA (panel A) or RVR (panel C) expression plasmids (+) in the presence of pRev-erb{alpha}WT reporter plasmid in 12-well plates and assayed for luciferase activity. Results shown are mean ± SD and were derived from three sets of independently transfected triplicates. Fold repression is expressed relative to repression obtained after cotransfection of pRev-erb{alpha}WT and pSG5 alone arbitrarily set to 1.0. Luciferase activity was determined after cotransfection of pRev-erb{alpha}WT (1 µg) and either the pSG5-hRev-erbA and pSG5-RVR wild-type and mutant plasmids (0.3 µg). B, Wild-type and mutant Rev-erb receptors were subcloned into the multiple cloning site of GALVP16 in frame and 3' of the GAL4/VP16 coding region. Cells were cotransfected into 12-well plates with pG5E1b-LUC reporter (1 µg) and GV-Rev chimeras (0.6 µg) and assayed for LUC activity. Results shown are mean ± SD and were derived from at least three independent transfections. Transcriptional repression is expressed relative to GAL4/VP16 alone.

 
To further understand the transcriptional properties of Rev-erbA and the LBD surface mutants in a more sensitive system, we examined whether the mutations in the LBD affected the ability of Rev-erbA to repress transactivation by the potent functional transactivator, GAL4VP16 (13, 14, 15). We subcloned the full-length wild-type and mutant Rev-erbA cDNAs into the GAL4VP16 expression vector and examined the effect on transactivation of the LUC reporter gene linked to GAL4 binding sites. A similar investigative approach utilizing the GAL4VP16 chimera has been previously used to analyze Rev-erbA and RVR (13, 14, 15). The GAL4VP16 protein, which contains the yeast GAL4 DBD and trans-activating domain of the herpes simplex virus VP16, is a potent transcriptional activator of the GAL4 binding sites linked to LUC.

Nine chimeric GAL4VP16-Rev-erbA expression plasmids were constructed, wild-type GALVP16-Rev-erb and eight mutants, GALVP16-Rev-erbA-W436A, GALVP16-Rev-erbA-F443A, GALVP16-Rev-erbA-V447A, GALVP16-Rev-erbA-V451A, GALVP16-Rev-erbA-R461A, GALVP16-Rev-erbA-V469A, GALVP16-Rev-erbA-K473A, and GALVP16-Rev-erbA-L606A. These mutants represented the range of responses observed in the in vitro/in vivo interaction studies (Figs. 4Go and 5Go) and the functional repression analysis (Fig. 6AGo). These were cotransfected with the reporter (pG5E1bLUC) into COS-1 cells, and the LUC activity was assayed (Fig. 6BGo). Full-length Rev-erb linked to the GALVP16 chimera very efficiently repressed (200-fold) the transcription of GALVP16 (Fig. 6BGo) as previously reported in this system (13, 14, 15). All the LBD surface mutants dramatically reduced the ability of Rev-erbA to repress GALVP16-mediated transactivation by 10- to 20-fold (Fig. 6BGo). In conclusion, these experiments further demonstrated that mutations in H3, loop 3–4, H4, and H11 significantly affected the function of the orphan receptors as transcriptional silencers.

We then investigated the impact of point mutations on RVR-mediated repression of the hRev-erbA{alpha} gene promoter (Fig. 6CGo). This promoter is far more efficiently repressed by RVR (than Rev-erbA) and more sensitive to the effect of mutations (24). Mutation of V410A, V414A, and K436A significantly reduced (~3- to 4-fold) the ability of RVR to repress luciferase expression driven by the Rev-erbA promoter (Fig. 6CGo). Mutation of W399A, F572A, and K573A reduced the ability of RVR by 2-fold to repress promoter expression. Mutation of F406A, V432A, and L569A K573A weakly reduced the ability of RVR to repress promoter expression by 1.5-fold. Mutation of R424A did not affect the ability of RVR to repress transcription.

The functional analysis of transcriptional repression by the native and mutant Rev-erb and RVR NRs supports the hypothesis that H3, loop 3–4, H4, and H11 are necessary for the silencing function of these orphan NRs (summarized in Tables 1Go and 2Go).


View this table:
[in this window]
[in a new window]
 
Table 1. hRev-erbA in Vitro/in Vivo Corepressor Interaction

 

View this table:
[in this window]
[in a new window]
 
Table 2. mRVR in Vitro/in Vivo Corepressor Interaction

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Transcriptional repression by the orphan NRs Rev-erbA and RVR is mediated by the corepressor N-CoR and its variants RIP13a and RIP13{Delta}1. N-CoR recruitment results in the assembly of a large multiprotein enzyme complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activity (36, 37, 38, 39, 40). This leads to 1) a reduced accessibility of DNA and 2) an alternate pathway of repression involving direct interactions with the key components of the basal transcriptional machinery [transcription factor IIB (TFIIB) and TBP-associated factors (TAFs)] that inhibit the initiation process (41, 42).

To resolve the structure and specificity of the orphan NR-corepressor interaction, and to address the issue "do all orphan NRs have ligands," we embarked on a three-dimensional analysis of Rev-erbA and RVR with an accompanying functional in vitro and in vivo interaction analysis with the corepressors. NR LBD structures known to date have confirmed the hypothesis of a highly conserved fold (16) and have prompted homology modeling studies in cases where the homology model could be tested by biochemical/pharmacological experiments, e.g. for DAX-1 (43), the estrogen receptor (ER) (44, 45), and the mineralocorticoid receptor (MR) (46). Here we have used a special method of homology modeling (31, 32) that avoids the bias introduced by the arbitrary initial conformation of the automatically replaced amino acids in other methods.

Rev-erbA and RVR May Lack Ligands
Ligand-dependent activation of transcription by NRs critically depends on the presence and integrity of H12 at the LBD C-terminus (25). Comparison of the crystal structures of the retinoid X receptor (RXR) apo-LBD and the RAR holo-LBD suggested that ligand binding caused receptor activation through repositioning of H12 (29). This was verified by solution studies with thyroid hormone receptor (TR) which demonstrated that this conformational change was required for corepressor release (47). Therefore, the absence of H12 in Rev-erbA/RVR together with their constitutive repressing activity suggest that these NRs have no endogenous ligand. In the homology models of the Rev-erbA and RVR LBDs presented here, the potential ligand cavity is filled up with side chains. This results from the presence of seven larger residues among the 21 homologous to the pocket-forming residues in RAR LBD (>21) and the shift of some secondary structure elements mainly caused by the absence of H12. The residual ligand-binding pocket has a probe-occupied volume of 16 Å3 in hRev-erbA{alpha} and cannot be detected in mRVR. One may ask whether an artifactual collapse may have occurred during the homology modeling because of ligand removal from the template structure. However, we have introduced harmonic restraints on the C{alpha} positions during the slow cooling step of the energy minimization to maintain the overall NR LBD fold. Furthermore, in the MR LBD homology model, which was built in a similar fashion using the hRAR{gamma} holo-LBD crystal structure as a template, a cavity with a probe-occupied volume of 469 Å3 was found, larger than that of the RAR LBD (418 Å3) and consistent with the size of aldosterone (303 Å3, compared with 278 Å3 for all-trans retinoic acid) (46). In fact, the numerous contacts between helices within the NR LBD fold probably prevent the collapse of the structure. In the case of PPAR{gamma}, the crystal structure of the apo-LBD shows a huge pocket (~1300 Å3), although not completely buried (28). In conclusion, although we cannot definitely conclude from homology modeling that Rev-erbA and RVR completely lack ligands, the present model structures establish that these orphans can adopt a low-energy conformation with no ligand-binding pocket, which probably represents the functional conformation since these proteins seem to act solely as transcriptional repressors.

There has been a controversial debate that has continued for many years in NR biology about whether "all the orphan receptors have ligands?", and was recently debated online by Giguère, Moore, and Wilson, and moderated by Lazar (48). Our study suggests that although the Rev-erbA/RVR receptors have a similar primary sequence, LBD structure, and common fold with respect to the classical NRs, the small size or absence of the pocket argues against the existence of a ligand. Furthermore, we suggest that other orphans exist that share the primary features and common fold of the classical NR LBDs, but probably lack a pocket or have insignificant ligand cavity sizes. Such orphans may represent the ancestral receptors, before the evolution of ligands and/or ligand binding (3). However, we do not preclude the identification or design of a synthetic ligand or regulatory molecule.

The Rev-erbA and RVR LBDs Encode Large Hydrophobic Surfaces
Our study demonstrated that the Rev-erbA/RVR LBD possesses a highly hydrophobic surface comprising H3, loop 3–4, H4, and H11. The main cause for the existence of such a surface is the shift of H11 toward H3 due to the absence of H12, allowing hydrophobic side chains from H3 and H11 to make numerous van der Waals contacts, resulting in H11 stabilization. Interestingly, the absence of H12, which is indicative of a repressing rather than a ligand-dependent activating function for a NR, would provide a simple way to create a permanent, hydrophobic interaction surface for a corepressor. Thus it is tempting to assume that the hydrophobic surface revealed by homology modeling is indeed the corepressor interacting surface.

Mutation of the Rev-erbA and RVR Hydrophobic Surfaces Impairs Corepressor Recruitment and Silencing of Transcription: H3, Loop 3–4, H4, and H11 Are Necessary
The mutagenesis coupled to biochemical and in vivo assays of corepressor binding, and the transcriptional repression assay of orphan NR function clearly supported the hypothesis that the hydrophobic surface comprised of H3, loop 3–4, H4, and H11 in Rev-erbA and RVR mediated corepressor recruitment and binding (summarized in Tables 1Go and 2Go). These data strongly reinforced our previous GAL4 hybrid studies that implicated H3, loop 3–4, H4, and H5 in transcriptional repression, and in vitro/in vivo interaction analysis, which demonstrated that H3 and H11 in RVR and Rev-erb were involved in corepressor recruitment and binding (14, 15). Our studies are in accord with the analysis of other NR-corepressor interactions, including the point mutation A483T in the surface-exposed loop between H3 and H4 in EcR, which disrupts corepressor binding (50), and the requirement of H11 of TR for SMRT recruitment and stabilization of the NR-corepressor interaction (51). Furthermore, point mutations in H1 of TR and RAR affect corepressor binding (17, 18). However, it should be noted that these latter mutations would disrupt the interactions of H1 with the LBD core and dislodge it from its native position, resulting in an impairment of the LBD function (16).

Our study has highlighted clear similarities and differences between Rev-erbA and RVR and the specificity of interaction with the variant corepressors.

A number of key similarities were observed with respect to the impact of similar mutation of corresponding Rev-erbA and RVR residues on the interaction with both corepressors (i.e. the N-CoR and RIP13{Delta}1 RIDs). For instance, the mutations V451A (H3) and V469A (H4) in Rev-erbA and the corresponding mutations V414A (H3) and V432A (H4) in RVR significantly reduced the ability of both Rev-erbA and RVR to interact with the two different corepressor isoforms in vivo and in vitro and to silence the Rev-erbA{alpha} promoter (see Tables 1Go and 2Go).

The corresponding mutations, F443A (H3) in Rev-erbA and F406A (H3) in RVR, do not affect the recruitment of the N-CoR RID. However, they significantly reduced the ability of both orphan NRs to interact with the RIP13{Delta}1 RID in vivo. Mutation R461A (loop 3–4) in hRev-erbA and the corresponding R424A mutation in RVR impacted on in vitro/in vivo interaction with the corepressors. Moreover, the mutation L606A (H11) in Rev-erbA and the corresponding mutation L569A (H11) in RVR significantly reduced the in vitro/in vivo interaction with the corepressors. In the case of these three sets of mutations the in vitro/in vivo protein-protein interaction studies are for the most part congruent; however, discrepancies exist in the repression of the Rev-erb promoter assay. It is our belief that this disparity is due to the weakness in the promoter repression assay, which is not particularly robust. This suggestion is supported by our demonstration that F443A, R461A, and L606A repress GAL4VP16-mediated transactivation by 5- to 20-fold (Fig. 6Go and Tables 1Go and 2Go).

The mutation F609A (H11) in Rev-erbA and the corresponding mutation F572A (H11) in RVR did not affect the ability of either Rev-erbA or RVR to interact with the corepressor in vivo and in vitro. However, these mutations significantly reduced the ability of these orphan NRs to silence transcription. This suggests these residues are involved in the transmission of the silencing signal to the transcriptional machinery.

On the other hand, there are some positions of subtle differences between Rev-erbA and RVR with respect to the impact of mutation on corepressor recruitment/association. Primarily, the W436A (H3) mutation in Rev-erbA affects interaction with the N-CoR and RIP13{Delta}1 RIDs, whereas the corresponding W399A (H3) mutation in RVR only affects RIP13{Delta}1 recruitment. However, they both consistently reduce the ability of the orphan NRs to silence gene expression. Similarly, the K473A mutation (H4) in Rev-erbA did not affect the ability of the receptor to recruit either corepressor; however, the corresponding mutation in RVR, K436A, significantly affected the recruitment of corepressors. Interestingly, both mutations in Rev-erbA and RVR compromised the ability of the receptors to repress transcription, respectively (Tables 1Go and 2Go).

The R610A (H11) mutation in Rev-erbA affects corepressor interactions with the N-CoR and RIP13{Delta}1 RIDs, yet has no effect on silencing, whereas the corresponding K573A (H11) mutation in RVR compromises corepressor recruitment and promoter silencing. In the models, R610 (Rev-erbA) and K573 (RVR) are engaged in different interactions, resulting in opposite side chain orientations (Fig. 3BGo): R610 is hydrogen bonded to the main chain carbonyl groups of T431 and A613 and forms a salt bridge with the C-terminal Q614 carboxylate, whereas K573 is only hydrogen bonded to the carbonyl group of L569 (RVR is shorter by one residue at its C terminus). This could explain the difference, not only between R610A and K573A, but also between W436A and W399A, since W399 in RVR is partially buried between K573 and E400 side chains, whereas W436 in Rev-erbA is exposed to the surface, R610 and E437 both pointing away from W436. However, it is also possible these differences arise from the proximity of the extra domain, which was not included in the model, and diverge widely between Rev-erbA and RVR.

Interestingly, seven mutations in hRev-erbA{alpha} (W436A, V447A, V451A, R461A, V469A, K473A, and L606A) significantly reduced by 10- to 20-fold, the ability of Rev-erbA to inhibit GAL4VP16-mediated transactivation of gene expression.

In conclusion, mutagenesis clearly supports the hypothesis that the hydrophobic surface comprised of H3, loop 3–4, H4, and H11 mediates the interaction with the corepressors, highlighting many similarities and some clear differences between Rev-erbA and RVR.

Related LBD Hydrophobic Surfaces Bind Both Corepressors and Coactivators
The NR/coactivator interface [TRß-GRIP-1/SRC-2, PPAR-{gamma}-SRC-1 and ER{alpha}-GRIP-1] has been documented by structural studies on the binding of coactivator LXXLL motifs to the LBD surface formed by H3-H4 and H12 (26, 27, 28) (summarized in Tables 1Go and 2Go). The present results thus suggest that corepressors and coactivators bind to overlapping surfaces of NR LBDs. The proposed differential binding of corepressors and coactivators is shown in Fig. 7Go. In classical NRs, H12 probably points away from the LBD core in the absence of ligand, as seen in the RXR apo-LBD crystal structure (52), and the corepressor is located on H3-H4 and H11 (Fig. 7AGo). The RXR apo-LBD crystal structure may not be prototypical for all apo-LBDs because H12 conformation probably has some dynamic character and exists under an equilibrium between several positions, and the conformation actually seen in the crystal structure is probably imposed by crystal packing. Nevertheless, it probably represents one among the accessible conformations in the apo state. Indeed, a recent molecular dynamics simulation study on ligand escape from the RAR LBD supports the idea that the apo state is structurally less constrained (53).



View larger version (51K):
[in this window]
[in a new window]
 
Figure 7. Differential Binding Of Corepressors and Coactivators to NR LBDs

A, In the absence of ligand, the corepressor is bound to the LBD (H3, H4, and H11), the activation helix H12 being away. This is exemplified by the RXR apo-LBD crystal structure (52 ), even though this crystallographic structure may represent only one of the possible conformations in the apo state (see text). The corepressor-interacting surface is proposed on the basis of the present study. B, Upon ligand binding, H12 is repositioned to a location that is no longer compatible with corepressor binding, which is therefore released. The newly formed AF-2 surface (H3, H4, and H12) can now recruit coactivators. This is exemplified by the RAR holo-LBD crystal structure (29 ), which indeed appears to be prototypical of the NR LBD canonical structure (16 25 ). The coactivator-interacting surface has been documented by the crystal structure of several complexes between a NR LBD and a coactivator-derived, LXXLL motif-containing peptide (26 27 28 ). [This figure was prepared with SETOR (57 ).]

 
It is not yet clear whether ligand binding directly causes corepressor dissociation, but this is suggested by the observation that a H12-truncated receptor binds the corepressor in the presence of the ligand (17). Upon ligand binding, H12 is brought against the LBD core, joining H3-H4 to form the complete AF-2 surface. This surface can no longer bind the corepressor but can now bind coactivators (Fig. 7BGo).

Thus it seems there is a mutual exclusion of both kinds of cofactors on the same LBD, hence the need of a ligand-dependent conformational switch (i.e. H12) acting as a lever to remove the corepressor. In the case of Rev-erbA/RVR, the absence of H12 and the proposed lack of ligand do not allow any conformational switch leading to corepressor release, accounting for the constitutive transcriptional repression.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Model Building
The starting point for homology modeling was the crystal structure of the hRAR{gamma} LBD (29). The hRAR{gamma}, hRev-erbA{alpha}, and mRVR sequences were aligned using the PileUp option of the GCG package (54) using a gap creation penalty of 4.0 instead of the default value of 3.0 to correctly align H1 in the three sequences (see Results) (Fig. 1Go). For the homology modeling of Rev-erbA, residues were replaced in O (55) according to the present alignment. Only residues 281–301 and 430–614 were taken into account as there was no way to model the extra domain reliably. Then an energy minimization was performed with X-PLOR (30) using a simulated annealing method for side chain building (31, 32). Briefly, the building procedure started from the main chain skeleton, and then the side chain atoms were built altogether starting from the C{alpha} atom positions by a simulated annealing protocol using packing and stereochemical restraints, avoiding the bias introduced by the arbitrary initial conformation of the automatically replaced amino acids; then the structure was refined by a slow cooling from 500 to 300 K followed by a Powell minimization. To maintain the LBD fold during the slow cooling, a harmonic restraint of 12.5 kcal/Å2 was applied to all C{alpha}’s and was then gradually released. The RVR model, comprising residues 243–263 and 393–576, was built in the same way starting from the Rev-erbA model, except that the harmonic restraint was not released during the slow cooling. This was justified by the fact that the sequence identity between Rev-erbA and RVR LBDs is very high (70%), especially at the level of the potential ligand-binding pocket (19 of the 21 residues homologous to the pocket-forming residues in RAR{gamma} are identical, see Fig. 1Go), with no insertion or deletion between H3 and H11, and thus it was reasonable to assume a strong conservation of the overall fold. The Ramachandran plots of the Rev-erbA and RVR models were calculated using PROCHECK (33), showing only 1 and 4 residues in disallowed regions, respectively. These residues all belong to loops, and their conformation was easily corrected by hand. The two models were then subjected to a final energy minimization (10 cycles of 100-step Powell minimizations without harmonic restraint). The final models contained no Ramachandran outliers. The Rev-erbA and RVR LBDs were superposed using the LSQ options of O (55), showing a rmsd of 0.5 Å over 205 C{alpha}’s. The probe-occupied cavities were determined and their volume computed with VOIDOO (34) using a probe radius of 1.4 Å (the probe sphere is rolled all over the protein cavity van der Waals surface and the contact points delimit the probe-occupied cavity). The atomic coordinates of the hRev-erbA{alpha} and mRVR LBD homology models have been deposited in the Protein Data Bank (PDB ID 1EF6 and 1EFJ, respectively).

Primer Sequences
The following primers were synthesized and used in a QuikChange site mutagenesis kit following manufacturer’s instructions with human Rev-erbA and mouse RVR cloned into the Stratagene expression vector, pSG5. Two primers were synthesized for the construction of each mutation as instructed by the manufacturer.

Primers Synthesized for the Point Mutations in Human Rev-erbA
W436A, GTG CAG GAG ATC GCG GAG GAT TTC TCC and GGA GAA ATC CTC CGC GAT CTC CTG CAC

F443A, C TCC ATG AGC GCC ACG CCC GCT G and C AGC GGG CGT GGC GCT CAT GGA G

V447A, C ACG CCC GCT GCA CGG GAG GTG G and C CAC CTC CCG TGC AGC GGG CGT G

V451A, CGG GAG GTG GCT GAG TTT GCC and GGC AAA CTC AGC CAC CTC CCG

R463A, C CCG GGC TTC GCA GAC CTT TCT CAG C and G CTG AGA AAG GTC TGC GAA GCC CGG G

V469A, G CAT GAC CAA GCC ACC CTG CTT AAG G and C CTT AAG CAG GGT GGC TTG GTC ATG C

K473A, GTC ACC CTG CTT GCG GCT GGC ACC and GGT GCCAGC CGC AAG CAG GGT GAC

L606A, G CAT TCC GAG AAG GCG CTG TCC TTC and GAA GGA CAG CGC CTT CTC GGA ATG C

F609A, GAG AAG CTG CTG TCC GCC CGG GTG GAC and GTC CAC CCG GGC GGA CAG CAG CTT CTC

R610A. CTG CTG TCC TTC GCG GTG GAC GCC CAG and CTG GGC GTC CAC CGC GAA GGA CAG CAG

Primers Synthesized for the Point Mutations in Mouse RVR
W399A, GGA CAT GAA ATC GCG GAA GAA TTT TCA ATG AG and CT CAT TGA AAA TTC TTC CGC GAT TTC ATG TCC

F406A, GG GAA GAA TTT TCA ATG AGT GCT ACC CCA GCA G and C TGC TGG GGT AGC ACT CAT TGA AAA TTC TTC CC

V410A, CC CCA GCA GCA AAA GAG GTG G and C CAC CTC TTT TGC TGC TGG GG

V414A, GCA GTA AAA GAG GTG GCG GAA TTT GC and GC AAA TTC CGC CAC CTC TTT TAC TGC

R424A, CCT GGC TTC GCA GAT CTG TCT CAG C and G CTG AGA CAG ATC TGC GAA GCC AGG

V432A, G CAT GAT CAG GCC AAT CTG TTA AAA GCT GG and CC AGC TTT TAA CAG ATT GGC CTG ATC ATG C

K436A, G GTC AAT CTG TTA GCA GCT GGG AC and GT CCC AGC TGC TAA CAG ATT GAC C

L569A, G CAC TCT GAG GAA GCC TTG GCC TTT AAA GTT CAT CC and GG ATG AAC TTT AAA GGC CAA GGC TTC CTC AGA GTG C

F572A, CTC TTG GCC GCT AAA GTT CAT CC and GG ATG AAC TTT AGC GGC CAA GAG

K573A, CTC TTG GCC TTT GCA GTT CAT CC and GG ATG AAC TGC AAA GGC CAA GAG

Mammalian Two-Hybrid Assays
Each well of a six-well plate of JEG-3 cells (60–70% confluence) was cotransfected with 3 µg pG5E1bLUC reporter, 1 µg GAL chimeras, and 1 µg VP16 chimeras in 1 ml of DMEM containing 5% charcoal-stripped FCS by the DOTAP (Roche Molecular Biochemicals) mediated procedure as described previously. After 24 h, the medium was replaced, and cells were harvested for the assay of chloramphenicol acetyltransferase (CAT) activity 36–48 h after transfection. Each transfection was performed at least three times to overcome the variability inherent in transfections (24).

Luciferase Assays
Luciferase activity was assayed using a Luclite kit (Packard Instruments, Meriden, CT) according to the manufacturer’s instructions. Briefly, cells were washed once in PBS and resuspended in 150 µl of phenol red-free DMEM and 150 µl of Luclite substrate buffer. Cell lysates were transferred to a 96-well plate, and relative luciferase units were measured for 5 sec in a Trilux 1450 microbeta luminometer (Wallac, Inc., Gaithersburg, MD) (24).

In Vitro Binding Assays
GST and GST-fusion proteins were expressed in Escherichia coli (BL21) and purified using glutathione-agarose affinity chromatography as described previously (32). The GST- fusion proteins were analyzed on 10% SDS-PAGE gels for integrity and to normalize the amount of each protein. The Promega Corp. TNT-coupled transcription-translation system was used to produce 35S-methionine-labeled RVR proteins that were visualized by SDS-PAGE. In vitro binding assays were performed with glutathione-agarose beads (Sigma) coated with approximately 500 ng of GST-fusion protein and 2–5 µl of 35S-methionine-labeled protein in 200 µl of binding buffer containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 5 µg ethidium bromide, and 100 µg BSA. The reaction was allowed to proceed for 1–2 h at 4 C with rocking. The affinity beads were then collected by centrifugation and washed five times with 1 ml of binding buffer without ethidium bromide and BSA. The beads were resuspended in 20 µl SDS-PAGE sample buffer and boiled for 5 min. The eluted proteins were fractionated by SDS-PAGE, the gel was treated with Amplify fluor (Amersham Pharmacia Biotech), dried at 70 C, and autoradiographed (24).


    ACKNOWLEDGMENTS
 
We thank Dino Moras for continued support and interest and for fruitful discussions. We are indebted to Michael Nilges for providing the scripts for the automated building procedure.


    FOOTNOTES
 
Address requests for reprints to: Dr. George E. O. Muscat, Institute for Molecular Bioscience, University of Queensland, Ritchie Research Laboratories, B402A Research Road, St. Lucia, 4072, Queensland, Australia.

This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, and the Ministère de l’Education Nationale, de la Recherche et de la Technologie (J.P.R.); and the National Health and Medical Research Council (NHMRC) of Australia (J.H., M.D., L.B., and G.E.O.M.). The Centre for Molecular and Cellular Biology is the recipient of an Australia Research Council (ARC) special research centre grant. G.E.O.M. is an NHMRC Senior Research Fellow.

Received for publication September 7, 1999. Revision received February 9, 2000. Accepted for publication February 10, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  2. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 86:835–839
  3. Escriva H, Safi R, Hänni C, Langlois MC, Stéhelin D, Capron A, Pierce R, Laudet V 1997 Ligand binding was acquired during evolution of nuclear receptors. Proc Natl Acad Sci USA 94:6803–6808[Abstract/Free Full Text]
  4. Miyajima N, Horiuchi R, Shibuya Y, Fukushige S, Matsubara K, Toyoshima K, Yamamoto T 1989 Two erbA homologs encoding proteins with different T3 binding capacities are transcribed from opposite DNA strands of the same locus. Cell 57:31–39[Medline]
  5. Lazar MA, Hodin RA, Cardona G, Chin WW 1990 Gene expression from the c-erbA{alpha}/Rev-erbA{alpha} genomic locus. Potential of alternative splicing by opposite strand transcription. J Biol Chem 265:12859–12863[Abstract/Free Full Text]
  6. Lazar MA, Hodin RA, Darling DS, Chin WW 1989 A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA{alpha} transcription unit. Mol Cell Biol 9:1128–1136[Medline]
  7. Munroe SH, Lazar MA 1991 Inhibition of c-erbA mRNA splicing by naturally occuring anti-sense RNA. J Biol Chem 266:22083–22086[Abstract/Free Full Text]
  8. Retnakaran R, Flock G, Giguère V 1994 Identification of RVR, a novel orphan nuclear receptor that acts as a negative transcriptional regulator. Mol Endocrinol 8:1234–1244[Abstract]
  9. Bonnelye E, Vanacker J-M, Desbiens X, Bègue A, Stehélin D, Laudet V 1994 Rev-erbß, a new member of the nuclear receptor superfamily, is expressed in the nervous system during chicken development. Cell Growth Diff 5:1357–1365[Abstract]
  10. Dumas B, Harding HP, Choi H-S, Lehmann KA, Lazar MA, Moore DD 1994 A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-erb. Mol Endocrinol 8:996–1005[Abstract]
  11. Harding HP, Lazar MA 1993 The orphan receptor Rev-ErbA{alpha} activates transcription via a novel response element. Mol Cell Biol 13:3113–3121[Abstract]
  12. Harding HP, Lazar MA 1995 The monomer-binding orphan receptor Rev-erb represses transcription as a dimer on a novel direct repeat. Mol Cell Biol 15:4791–4802[Abstract]
  13. Downes M, Carozzi A, Muscat GEO 1995 Constitutive repression of the orphan receptor, Rev-erbA{alpha}, inhibits muscle differentiation and abrogates the expression of the myoD gene family. Mol Endocrinol 9:1666–1678[Abstract]
  14. Downes M, Burke LJ, Muscat GEO 1996 Transcriptional repression by Rev-erbA{alpha} is dependent on the signature motif and helix 5 in the ligand binding domain. Nucleic Acids Res 24:3490–3498[Abstract/Free Full Text]
  15. Burke L, Downes M, Carozzi A, Giguère V, Muscat GEO 1996 Transcriptional repression by the orphan receptor RVR/Rev-erb-ß is dependent on the signature motif and helix 5 in the E region: functional evidence for a biological role of RVR in myogenesis. Nucleic Acids Res 24:3481–3489[Abstract/Free Full Text]
  16. Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nat Struct Biol 3:87–94[Medline]
  17. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  18. Hörlein AJ, Näär AN, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Söderström M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  19. Sande S, Privalsky ML 1996 Identification of TRACs (T3 receptor-associating cofactors), a family of cofactors that associate with, and modulate the activity of, nuclear hormone receptors. Mol Endocrinol 10:813–825[Abstract]
  20. Downes M, Burke LJ, Bailey PJ, Muscat GEO 1996 Two receptor interaction domains in the corepressor, N-CoR/RIP13, are required for an efficient interaction with Rev-erbA{alpha} and RVR: physical association is dependent on the E region of the orphan receptors. Nucleic Acids Res 24:4379–4386[Abstract/Free Full Text]
  21. Zamir I, Harding HP, Atkins GB, Hörlein A, Glass CK, Rosenfeld MG, Lazar MA 1996 A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains. Mol Cell Biol 16:5458–5465[Abstract]
  22. Seol W, Mahon MJ, Lee Y-K, Moore DD 1996 Two receptor interaction domains in the nuclear hormone receptor corepressor RIP13/N-CoR. Mol Endocrinol 10:1646–1655[Abstract]
  23. Zamir I, Zhang J, Lazar M 1997 Stoichiometric and steric principles governing repression by nuclear hormone receptors. Genes Dev 11:835–846[Abstract]
  24. Burke LJ, Downes M, Laudet V, Muscat GEO 1998 Identification and characterization of a novel corepressor interaction region in RVR and Rev-erbA{alpha}. Mol Endocrinol 12:248–262[Abstract/Free Full Text]
  25. Moras D, Gronemeyer H 1998 The nuclear receptor ligand-binding domain: structure and function. Curr Opin Cell Biol 10:384–391[CrossRef][Medline]
  26. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[Medline]
  27. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3343–3356[Abstract/Free Full Text]
  28. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  29. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-gamma ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  30. Brünger AT 1992 X-PLOR Version 3.1. Yale University Press, New Haven, CT
  31. Nilges M, Brünger AT 1991 Automated modeling of coiled coils: application to the GCN4 dimerization region. Protein Eng 4:649–659[Abstract]
  32. Nilges M, Brünger AT 1993 Successful prediction of the coiled coil geometry of the GCN4 leucine zipper domain by simulated annealing: comparison to the X-ray structure. Proteins 15:133–146[Medline]
  33. Laskowski RA, MacArthur MW, Moss DS, Thornton JM 1993 PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291[CrossRef]
  34. Kleywegt GJ, Jones TA 1994 Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr D 50:178–185[CrossRef]
  35. Henttu PM, Kalkhoven E, Parker MG 1997 AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors. Mol Cell Biol 17:1832–1839[Abstract]
  36. Xu L, Glass CK, Rosenfeld MG 1999 Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Gen Dev 9:140–147[CrossRef][Medline]
  37. Wolffe AP, Hayes JJ 1999 Chromatin disruption and modification. Nucleic Acids Res 27:711–720[Abstract/Free Full Text]
  38. Wollfe AP 1997 Sinful repression. Nature 387:16–17[CrossRef][Medline]
  39. Archer SY, Hodin RA 1999 Histone acetylation and cancer. Curr Opin Gen Dev 9:171–174[CrossRef][Medline]
  40. Jacobson S, Pillus L 1999 Modifying chromatin and concepts in cancer. Curr Opin Gen Dev 9:175–184[CrossRef][Medline]
  41. Muscat GEO, Burke L, Downes M 1998 The corepressor N-CoR and its variants RIP13a and RIP13{Delta}1 directly interact with the basal transcription factors, TFIIB, TAFII32 and TAFII70. Nucleic Acids Res 26:2899–2907[Abstract/Free Full Text]
  42. Wong CW, Privalsky ML 1998 Transcriptional repression by the SMRT-mSin3 corepressor: multiple interactions, multiple mechanisms and a potential role for TFIIB. Mol Cell Biol 18:5500–5510[Abstract/Free Full Text]
  43. Lalli E, Bardoni B, Zazopoulos E, Wurtz JM, Strom TM, Moras D, Sassone-Corsi P 1997 A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita. Mol Endocrinol 11:1950–1960[Abstract/Free Full Text]
  44. Maalouf GJ, Xu W, Smith TF, Mohr SC 1998 Homology model for the ligand-binding domain of the human estrogen receptor. J Biomol Struct Dyn 15:841–851[Medline]
  45. Wurtz JM, Egner U, Heinrich N, Moras D, Mueller-Fahrnow A 1998 Three-dimensional models of estrogen receptor ligand binding domain complexes, based on related crystal structures and mutational and structure-activity relationship data. J Med Chem 41:1803–1814[CrossRef][Medline]
  46. Fagart J, Wurtz JM, Souque A, Hellal-Levy C, Moras D, Rafestin-Oblin ME 1998 Antagonism in the human mineralocorticoid receptor. EMBO J 17:3317–3325[Abstract/Free Full Text]
  47. Lin B, Hong SH, Krig S, Yoh S, Privalsky ML 1997 A conformational switch in nuclear hormone receptors is involved in coupling hormone binding to corepressor release. Mol Cell Biol 17:6131–6138[Abstract]
  48. Giguère V, Lazar M, Moore DD, Willson T 1997 Biomednet-HMS Beagle, vol 1, no. 2, Feb 17, 1997
  49. Vivat V, Zechel C, Wurtz JM, Bourget W, Kagechika H, Umemiya H, Shudo K, Moras D, Gronemeyer H, Chambon P 1997 A mutation mimicking ligand-induced conformational change yields a constitutive RXR that senses allosteric effects in heterodimers. EMBO J 16:5697–5709[Abstract/Free Full Text]
  50. Tsai CC, Kao HY, Yao TP, McKeown M, Evans RM 1999 SMRTER, a drosophila nuclear receptor coregulator, reveals that EcR-mediated repression is critical for development. Mol Cell 4:175–186[Medline]
  51. Li H, Leo C, Schroen DJ, Chen JD 1997 Characterization of receptor interaction and transcriptional repression by the corepressor SMRT. Mol Endocrinol 11:2025–2037[Abstract/Free Full Text]
  52. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR{alpha}. Nature 375:377–382[CrossRef][Medline]
  53. Blondel A, Renaud JP, Fischer S, Moras D, Karplus M 1999 Retinoic acid receptor: a simulation analysis of retinoic acid binding and the resulting conformational changes. J Mol Biol 291:101–115[CrossRef][Medline]
  54. Genetics Computer Group 1994 Program Manual for the Wisconsin Package, Version 8. Genetics Computer Group, Madison, WI
  55. Jones TA, Zou JY, Cowan SW, Kjeldgaard M 1991 Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47:110–119[CrossRef][Medline]
  56. Nicholls A 1993 GRASP: Graphical Representation and Analysis of Surface Properties. Columbia University, New York
  57. Evans SV 1993 SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graphics 11:134–138[CrossRef][Medline]
  58. Adelmant G, Bègue A, Stéhelin D, Laudet V 1996 A functional Rev-erb{alpha} responsive element located in the human Rev-erb{alpha} promoter mediates a repressing activity. Proc Natl Acad Sci USA 93:3553–3558[Abstract/Free Full Text]