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
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ABSTRACT |
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INTRODUCTION |
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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 455488 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 H3H5.
Ligand-independent repression of transcription by Rev-erbA and RVR is
mediated by the nuclear receptor corepressor N-CoR and its variants
RIP13a and RIP131 (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
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 407418) and
domain Y (aa 602614) (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. 1). 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
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
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
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.
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RESULTS |
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The template for homology modeling was the crystal structure of the
hRAR LBD (29). The extra domains were not included so the homology
models comprised residues 281301 and 430614 for Rev-erbA, and
residues 243263 and 393576 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. 2A
) was
obtained manually by optimally superposing the central H4H5 helices
only (residues 465487/428450/256278, hRev-erbA
/mRVR/hRAR
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).
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Indeed, in the Rev-erbA/RVR models, the ligand cavity is occupied by
side chains, with no room left for a potential ligand (Fig. 2B). This
results from both the presence of bigger side chains at positions
homologous to the pocket-forming residues in RAR
and the shift of
some secondary structure elements (Figs. 1
and 2
). 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
/hRev-erbA
/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
(Fig. 2
, B and
C). At the center, the volume of the retinoic acid cavity in hRAR
is
filled up by the F484/447, L498/461, M513/A476, L516/479, and H602/565
side chains (Fig. 2C
). Calculations with VOIDOO (34) indicate that the
residual ligand-binding pocket has a probe-occupied volume of 16
Å3 in hRev-erbA
(compared with 418
Å3 in hRAR
), 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. 1
): 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. 2B
).
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 34, 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 34, V469/432
and K473/436 in H4, and L606/569, F609/572, and R610/573 in H11
(hRev-erbA/mRVR numbering) (Fig. 3
).
Comparison of the Rev-erbA/RVR and RAR C
traces (Fig. 2A
) 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. 2B
and 3B
).
By contrast, H3 and H11 in hRAR
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
, respectively. Thus, it seems that H3 and H11 in
Rev-erbA form together a continuous hydrophobic surface, extended by
side chains from loop 34 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. 2B
and 3B
).
Nevertheless, H3 and H11 still form together a hydrophobic surface,
very similar to that in Rev-erbA.
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In Vitro Mutational Analysis of the Hydrophobic Surface
in the Rev-erbA LBD: H3, Loop 34, 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 34, 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, RIP131. We measured and examined the
interaction between Rev-erbA and RVR, and the RIDs from N-CoR and
RIP13
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. 1
and 3B
).
The mutations in hRev-erbA
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 RIP131. The RIDs from N-CoR and RIP13
1 are
similar. However, the RID from RIP13
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
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
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 RIP131 RIDs.
Glutathione agarose-immobilized GST-N-CoR and RIP131 RIDs were
tested for direct interaction with in vitro
35S-radiolabeled native and mutant Rev-erbA
proteins (Fig. 4
). The GST-N-CoR/RIP13a
and RIP13
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
34 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.
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In Vitro Mutational Analysis of the Hydrophobic Surface
in the RVR LBD: H3, Loop 34, H4, and H11 Are Directly Involved in
Corepressor Binding
We then investigated the potential of various single-point
mutations in H3, loop 34, H4, and H11 of RVR [in the context of the
full-length receptor] to interact with the N-CoR and RIP131 RIDs in
the GST-pulldown assay (Fig. 4
). Interestingly, V410A and V414A in H3
of RVR had minor and significant effects, respectively, on the
interaction with the N-CoR and RIP13
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
1 RIDs).
Curiously, R424A in loop 34 significantly increased binding to the
N-CoR RID, but not to the RIP131 RID. Mutations V432A and K436A in
H4 did not effect binding to the N-CoR RID. However, they significantly
affected binding to the RIP13
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
1 RID.
In summary, these in vitro biochemical results suggest that H3, loop 34, 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 34, 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 34, 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 RIP131 RIDs, which are very similar,
except for an internal deletion of 120 amino acids in RIP13
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
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
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. 5A
).
We observed that mutations W436A (H3), V451A (H3), R461A (loop 34),
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
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
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
1 RIDs (Fig. 5A
).
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These results suggest that the functional interaction, in vivo, is dependent on the hydrophobic surface of the Rev-erbA LBD that encompasses H3, loop 34, 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 34, 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 34, H4, and H11 Are Directly Involved in
Corepressor Binding
We then investigated the potential of various single-point
mutations in the H3, loop 34, H4, and H11 of RVR [in the context of
the full-length receptor] to interact with the N-CoR and RIP131
RIDs in the mammalian two-hybrid assay. The chimeric construct
consisting of the yeast GAL4 DBD fused to the N-CoR and RIP13
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. 5C
). However, the interaction of RVR
with the RIP13
1 RID was less robust; we observed that mutation F406
in H3 dramatically reduced the corepressor interaction, and mutations
V414A (H3), R424A (loop 34), V432A (H4), K436A (H4), and L569A (H11)
significantly inhibited the interaction with the RIP13
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
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. 5B
). Additionally, a lack of RVR degradation products
indicates that the wild-type and mutant proteins have similar
stabilities (Fig. 5B
).
The analysis of the interaction between the corepressors and RVR suggests that the hydrophobic surface of the RVR LBD that encompasses H3, loop 34, 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 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 34, 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
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
represses
transcription of its own promoter by approximately 3-fold (24, 58). The
mutations in hRev-erbA
at positions F443A and V447A ablated the
ability of Rev-erb to silence promoter expression (Fig. 6A
). 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.
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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. 4 and 5
) and
the functional repression analysis (Fig. 6A
). These were cotransfected
with the reporter (pG5E1bLUC) into COS-1 cells, and the LUC activity
was assayed (Fig. 6B
). Full-length Rev-erb linked to the GALVP16
chimera very efficiently repressed (200-fold) the transcription of
GALVP16 (Fig. 6B
) 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. 6B
).
In conclusion, these experiments further demonstrated that mutations in
H3, loop 34, 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 gene promoter (Fig. 6C
). 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. 6C
). 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 34,
H4, and H11 are necessary for the silencing function of these orphan
NRs (summarized in Tables 1 and 2
).
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DISCUSSION |
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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 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
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
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
, 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 34, 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 34,
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 34, H4, and H11 in Rev-erbA
and RVR mediated corepressor recruitment and binding (summarized in
Tables 1 and 2
). These data strongly reinforced our previous GAL4
hybrid studies that implicated H3, loop 34, 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
RIP131 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
promoter (see
Tables 1
and 2
).
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 RIP131 RID in vivo. Mutation R461A (loop 34) 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. 6
and Tables 1
and 2
).
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 RIP131 RIDs,
whereas the corresponding W399A (H3) mutation in RVR only affects
RIP13
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 1
and 2
).
The R610A (H11) mutation in Rev-erbA affects corepressor interactions
with the N-CoR and RIP131 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. 3B
): 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 (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 34, 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--SRC-1
and ER
-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 1
and 2
). 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. 7
. 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. 7A
). 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).
|
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.
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MATERIAL AND METHODS |
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Primer Sequences
The following primers were synthesized and used in a QuikChange
site mutagenesis kit following manufacturers 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 (6070% 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 3648 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 manufacturers
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 25 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 12 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 lEducation 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.
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REFERENCES |
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