From the Metabolic Research Unit Department of
Medicine and the ** Department of Biochemistry and Biophysics,
University of California, San Francisco, California 94143 and the
§ Department of Pharmaceutical Sciences, University of
Brasilia, DF 70910-900, Brazil
Received for publication, November 8, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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Thyroid hormone receptors (TRs) bind as
homodimers or heterodimers with retinoid X receptors (RXRs) to DNA
elements with diverse orientations of AGGTCA half-sites. We
performed a comprehensive x-ray crystal structure-guided mutation
analysis of the TR ligand binding domain (TR LBD) surface to map
the functional interface for TR homodimers and heterodimers with RXR in
the absence and/or in the presence of DNA. We also identified the
molecular contacts in TR LBDs crystallized as dimers. The results show
that crystal dimer contacts differ from those found in the functional
studies. We found that identical TR LBD residues found in helices 10 and 11 are involved in TR homodimerization and heterodimerization with
RXR. Moreover, the same TR LBD surface is operative for dimerization with direct repeats spaced by 4 base pairs (DR-4) and with the inverted palindrome spaced by 6 base pairs (F2), but not
with TREpal (unspaced palindrome), where homodimers appear to be simply two monomers binding independently to DNA. We also demonstrate that
interactions between the TR and RXR DNA binding domains stabilize TR-RXR heterodimers on DR-4. The dimer interface can be
functional in the cell, because disruption of key residues impairs
transcriptional activity of TRs mediated through association with RXR
LBD linked to GAL4 DNA-binding domain.
Thyroid hormone receptors
(TRs)1 are members of the
nuclear receptor superfamily, which includes receptors for steroid
hormones, vitamins, retinoids, prostaglandins, fatty acids, and orphan
receptors for which no ligands are known (1-4). These receptors are
modular transcription factors that bind to specific sequences in
promoters of target genes. They bind DNA as monomers, homodimers, or
heterodimers through the DNA binding domain (DBD) (1, 5). Steroid
receptors bind as homodimers to two half-sites of a DNA palindrome. In
contrast, TRs, retinoic acid receptors (RARs), vitamin D receptors
(VDRs), peroxisome proliferator-activated receptors (PPARs), and
several orphan receptors bind as heterodimers with retinoid X receptors (RXRs) to direct repeats (DRs) of the AGGTCA half-site (1) but also
bind to elements oriented as palindromes or inverted palindromes (1).
Binding specificity of these receptors is determined by spacing between
the half-sites, because PPARs, VDRs, TRs, and RARs bind preferentially
to DRs spaced by 1, 3, 4, or 5 nucleotides, respectively (1). Unlike
PPARs, VDRs, and RARs, TRs also bind DNA as monomers and as homodimers
to DRs and to inverted palindromes spaced by 4-6 nucleotides (F2) (1,
6, 7).
Current concepts of the dimerization and heterodimerization interfaces
for nuclear receptors are based on mutational and x-ray crystal
structural data. The mutation data for LBDs are mostly limited to TR,
RAR, VDR, and RXR. Little is known from mutation studies with the
steroid receptors, because only few studies report single mutations in
the LBDs that disrupt dimerization (8). This is due in part to the fact
that the DBDs and amino-terminal fragments of these receptors
contribute substantially to stabilization of the dimer (9, 10).
Data for the TR, RXR, RAR, and VDR LBD dimerization have come from
point mutations and deletions. These studies have identified an area
encompassing about 40 amino acids in helices 10 and 11 (H10 and H11)
that appears to mediate formation of RXR, and TR homodimers, and
RAR-RXR, TR-RXR, and VDR-RXR heterodimers (11-15). However, this
interpretation was based on results with mutations of residues placed
in the interior core structure of the LBD, such as with the leucines in
H10 and H11 in the so-called heptad repeats (16). Thus, these and the
deletion mutations may disrupt receptor folding, complicate the
interpretation of results, and, consequently, not allow for a
definition of the actual interface or specific residues involved in
forming homodimers or heterodimers.
Mutation data have also suggested that the region and residues within
the TR LBD that form homodimers are distinct from those that form
heterodimers. For example, changing leucines to arginines in H11 of
chicken TR Several investigators demonstrated that TR-RXR heterodimers interact in
solution through a LBD heterodimer interface. It has been proposed that
these heterodimers bind DR-4 elements by initial formation in solution
of the LBD-LBD interface, which leads, upon DNA binding, to the
formation of a second interface involving the second zinc finger of the
RXR DBD and the first zinc finger of the TR DBD (19-22). Indeed,
crystallographic studies of isolated TR-RXR DBDs bound to DR-4
supported the existence of a DBD heterodimer interface in which the RXR
occupies the 5' half-site, whereas TR occupies the 3' half-site (23).
However, there is no functional evidence that DBD surfaces participate
in complexes formed by full-length TR homodimers or TR-RXR heterodimers
bound to DR-4 or other elements. Also, if DBD surfaces participate in
dimerization, it is unclear if the LBD and DBD surfaces form
sequentially, nor is it known whether a LBD surface is used by TR
homodimers to bind any DNA element. To date, no x-ray crystal
structural data are available for TR LBD homodimers or heterodimers
bound to DNA, and no comprehensive mutation analyses are available that
locate the homodimer or heterodimer TR LBD interface.
RXR The TR LBD structure reported to date is formed by TR monomers. Thus it
is not apparent which TR residues participate in homodimer or
heterodimer interactions and whether they vary depending on the nature
of the TRE. Furthermore, structural data do not demonstrate functional
TR interactions. In the studies reported here we performed a
comprehensive x-ray crystal structure-guided mutation analysis of the
TR LBD surface to map the functional interface for TR homodimers and
heterodimers with RXR in the absence and/or in the presence of DNA.
This approach employed the x-ray crystal structures of the TR-RXR DBDs
and TR LBD (16, 23) and was previously used by us for definition of a
TR coactivator-binding surface (34). We also identified the molecular
contacts in TR LBDs crystallized as dimers. The results show that
crystal dimer contacts differ from those found in the functional
studies. We found that identical TR LBD residues are involved in TR
homodimerization and heterodimerization with RXR. Further, the same LBD
surface is operative irrespective of the orientation and spacing of the
half-sites for DR and inverted palindromic elements, but not for the
TREpal homodimers, which appear to be simply two monomers binding to
DNA. We also demonstrate that interactions between the DBDs stabilize
TR-RXR heterodimers on DR-4. The dimer interface can be functional in
the cell, because disruption of key residues impairs transcriptional
activity of TRs mediated through association with RXR LBD linked to
GAL4 DNA-binding domain.
Construction of TR Mutants--
TR mutants were created by
ligating double-stranded oligonucleotides encoding the mutant sequence
into the pCMX vector that encodes the full-length 461 amino acid
hTR
The relative affinity was determined by dividing the wild type by the
mutant Kd. The 62 mutants included and their relative affinities are: E217R (91%), W219K (not done (ND)), E227R (109%), K242E (68%), F245K (ND), D249R (ND), V256R (ND), L266K (ND),
E267R (87%), H271R (91%), T277R (7%), T281R (94%), V284R (105%),
D285A (68%), K288A (61%), M292K (ND), C294K (94%), E295R (150%),
C298A (65%), C298R (44%), E299A (139%), I302A (91%), I302R (99%),
L305I (ND), K306A (5%), K306E (7%), C309A (ND), V319K (ND), E324R
(ND), M334K (ND), V348R (ND), D351A (ND), D355A (ND), M358A (ND), D382R
(86%), P384R (121%), A387R (80%), V389A (59%), E390R (140%), E393R
(109%), L400R (70%), H413R (81%), V414K (ND), H416R (113%), P419R
(152%), L422R (69%), M423R (174%), T426R (48%), R429A (55%), M430R
(107%), A433R (ND), C434R (124%), S437R (65%), L440R (129%), V444R
(66%), T448R (282%), E449R (26%), P453E (32%), L454R (12%), L456R
(13%), E457K (38%), V458M (ND).
A TR DBD mutant was created by changing the Asp177, known
to interact with RXR in crystallographic studies, to Ala. A RXR DBD mutant was made by changing all Arg residues previously shown in
crystallographic studies to be involved in dimerization with the TR to
Ala (R172A, R182A, and R186A, Ref. 23). We also found that this RXR
mutant had three additional DBD changes (T168P, T170I, and Y193A) that
did not interfere with RXR binding to DNA. An RXR LBD mutant was made
by changing leucines 419 and 420 into arginines, positions analogous to
TR mutants defective in dimerization (see "Results").
Crystallization
hTR rTR Structural Analysis
hTR rTR Glutathione S-Transferase Assay
For these experiments full-length hRXR Gel Shift Binding Assay
Binding of full-length hTR Cell Culture, Electroporation, and Luciferase Assays
CV1 cells were maintained and subcultured in media DME H-21 4.5 g/l glucose, containing 10% newborn bovine serum, 2 mM
glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.
Transfection procedures were described previously (37). Briefly, cells
were collected and resuspended in Dulbecco's phosphate-buffered
saline (0.5 ml/1.5 × 107 cells) containing 0.1%
dextrose, and typically 10 µg of reporter plasmid and 1 µg of
expression vector. Cells were electroporated at 350 V and 960 microfarads, transferred to fresh media, and then plated in 6-well
plates. After incubation for 24 h at 37 °C with ethanol, or 100 nM T3, the cells were collected and the pellets
were solubilized by addition of 100 µl of 0.25 M
Tris-HCl, pH 7.6 containing 0.1% Triton X-100. For each transfection,
1 µg of the pCMVhTR Models of the TR-RXR Heterodimer
These models were derived using the known structures of the TR
(27) and the hRAR TR LBDs Form Crystals with Diverse Intermolecular
Contacts--
Crystal forms of LBD fragments of the TR
In TR Scanning Mutations Define a LBD Interface for TR-RXR Association in
Solution--
Because the hydrophobic contacts described above may
reflect forces operative during crystallization and not represent a
functional dimerization surface, we mapped the TR LBD surface that
participates in dimer formation by mutating the TR LBD surface. These
mutations were introduced ~7 Å apart to scan the TR LBD area
involved in protein-protein interactions (34). As described under the
"Materials and Methods," these mutations generally maintained
overall receptor integrity and functions largely unrelated to dimer or
heterodimer formation such as ligand and coactivator binding.
Sites important for TR-RXR interactions were first determined by
examining binding of TRs to GST·RXR in pull-down assays. Wild-type
hTR The LBD Surface That Forms TR-RXR Heterodimers in Solution also
Forms TR Homodimers on DR-4 and F2 but Not on TREpal--
Because
homodimers with full-length TRs are not observed in solution, we next
sought to define the TR surface used by TR homodimers on DNA. We first
used the DR-4 binding site, because this is the most commonly described
TRE. As shown in Fig. 3A, we
found that a subset of the mutants that inhibited formation of TR-RXR
heterodimers in solution also blocked TR homodimer formation on DR-4.
Thus, E393R and L400R in H10 (lanes 5 and 7,
respectively) and P419R, L422R, and M423R in H11 (lanes 13,
15, and 17) nearly abolished formation of TR
homodimers on DR-4. The mutants T426R and M430R in H11 that impaired
formation of TR-RXR heterodimers in solution had minimal influences on
TR homodimer formation (Fig. 3A, lanes 19 and
21). No other TR mutants of the 50 tested spanning helices 1, 3, 5, 8, 9, and 12 were defective in homodimer assembly on DR-4
(data not shown).
Because inverted palindrome DNA sequences have the highest capacity for
binding TR homodimers (6), we used the F2 palindrome to examine whether
the TR LBD surface that forms homodimers on F2 coincides with that
mapped for homodimers on DR-4 and for TR-RXR heterodimers in solution.
Fig. 3B shows that this is the case because L400R in H10 and
L422R and M423R in H11 (lanes 7, 15, and
17) disrupted homodimers with F2. However, in contrast to DR-4, the E393R and P419R mutants (lanes 5 and
13) partially disrupt homodimers on F2. These results
demonstrate that the essential residues required for forming TR LBD
homodimers with DR-4 or F2 are Leu400 in H10 and
Leu422 and Met423 in H11. However, a larger
group of residues flanking this critical core of amino acids is
required for forming TR homodimers with DR-4 compared with F2.
We also asked if the surface used for TR homodimers on DR-4 and F2 and
for TR-RXR heterodimers in solution is also operative for forming TR
homodimers on TREpal. None of these mutants (Fig. 3C) or
other surface TR mutants tested (data not shown) blocked formation of
TR homodimers on TREpal. These data suggest that TRs bound to TREpal
are simply two monomers rather than a homodimer.
Taken together, these results show that the TR LBD surface involved in
formation of TR-RXR heterodimers in solution and TR homodimers with
DR-4 or F2 is similar and is not involved in TRs bound to TREpal. This
surface contains a critical core of hydrophobic amino acids comprised
of Leu400 in H10 and Leu422 and
Met423 in H11 that are required for all types of TR dimeric
interactions. In H11, the residues flanking Leu422 and
Met423 appear to be important for homodimers on DR-4 and
for heterodimers in solution. These findings suggest that the dimer
surface is centered on a set of critical hydrophobic residues, but that
it is flexible and extends to adjacent residues (Fig.
3D).
The Same LBD Interface Mediates Formation of TR Homodimers and
TR-RXR Heterodimers on DNA, but Heterodimers Are More Stable because of
DBD-DBD Interactions--
The mutations were examined for effects on
TR-RXR heterodimer formation on DNA. None of the TR LBD single mutants
that impaired formation of TR homodimers on DR-4 (see Fig.
3A) or an additional double mutant L422R/M423R
containing two residues on the dimerization surface (data not shown)
disrupted TR-RXR heterodimers on DR-4. However, L422R weakened
formation of TR-RXR heterodimers on F2 (Fig. 3B, lane
16) and abolished them on TREpal (Fig. 3C, lane 16). Because Leu422 in the center of the surface is
critical for TR-RXR heterodimers in solution and TR homodimers on DR-4
and F2, it is likely that the TR-RXR heterodimer surface on F2, TREpal,
and even DR-4 is also similar but that TR-RXR heterodimers are more stable.
The greater stability of TR-RXR heterodimers on DR-4 could be in part
because of DBD-DBD contacts as observed in the x-ray crystal structure
of TR-RXR isolated DBDs (without LBDs) on DR-4 (23). However, the
greater resistance to mutational disruption of the DR-4 bound TR-RXR
heterodimers as compared with TR-TR homodimers could be because of the
fact that only the TR subunit of the heterodimer pair contained a
mutation whereas with TR-TR homodimers both subunits are mutated. To
address this issue, we assayed TR-RXR heterodimer formation where both
the TR and RXR subunits are mutated. For these experiments, we replaced
two leucines, Leu419 and Leu420, with arginines
in the RXR LBD, which are residues analogous to the TR double mutant
L422R/M423R that did not disrupt TR-RXR heterodimers on DR-4. As shown
in Fig. 4A, the RXR
L419R/L420R mutant markedly inhibited heterodimer formation with
wild-type TR on DR-4 (lane 4). Mixing RXR L419R/L420R with
TR L422R (Fig. 4A, lane 8) or L422R/M423R (data
not shown) did not further disrupt heterodimers on DR-4. These findings
indicate that those mutations in the LBDs of both subunits can reduce
heterodimer formation on DR-4 and suggest that the RXR LBD dimer
interface is similar to the one in the TR LBD. These findings together
with the known structures of the TR (27) and the hRAR
We also investigated if DBD-DBD interactions participate in TR-RXR
heterodimers on DR-4 by using TR and RXR DBD mutations based on the
crystal structure of the TR-RXR DBD dimers (23). The TR DBD mutant
replaced the aspartic acid 177 with alanine and the RXR DBD mutant
replaced the three arginines 172, 182, and 186 with alanines. The TR
D177A formed efficient homodimers and heterodimers with either the
wild-type RXR or the RXR DBD mutant on DR-4 (Fig. 4A,
compare lanes 1-3 to 9-11) indicating that
these DBD mutations per se do not disrupt formation of
homodimers or heterodimers on DR-4. In contrast, heterodimer formation
was completely abolished by mixing the TR D177A with the RXR
L419R/L420R LBD mutant (Fig. 4A, lane 12).
Similarly, TR-RXR heterodimers did not form (Fig. 4A, lane
7) when the RXR DBD mutant was mixed with the TR LBD mutants L422R
(that formed efficient heterodimers with the wild-type RXR; Figs.
3A, lane 16 and 4A, lane 6)
or L422R/M423R (data not shown). By contrast, the RXR DBD mutant did
not disrupt heterodimers with L422R on the F2 DNA (data not shown).
Collectively, the data show that LBD interactions are dominant in
formation of TR homodimers and heterodimers on DR-4 because disruptions in LBD, but not in DBD interactions, impair TR dimer formation. These
results also indicate that DBD interactions stabilize TR-RXR heterodimers on DR-4, because DBD mutations can enhance the effects of
LBD mutations. The data provide additional evidence that the LBD
surfaces that form TR-RXR heterodimers on DR-4 are similar to those
utilized by TR-RXR heterodimers in solution and by TR homodimers on
DR-4.
Mutation of TR Hydrophobic Residues in Helix 11 Impairs
Transactivation in Vivo When RXR Is Linked to a Heterologous
DBD--
The TR LBD single mutants that impair assembly of TR
homodimers on DNA and TR-RXR heterodimers in solution were assessed for their abilities to induce ligand-mediated transcription in cultured cells in a context where the interactions occur only through the LBDs,
and the TR is not bound to DNA. We cotransfected GAL·RXR with TRs in
CV-1 cells. In the presence of wild-type TR or any of the mutants that
failed to impair the TR-RXR association in solution (E390R, H416R),
T3 stimulated 25- to 48-fold the reporter gene driven by
five GAL binding sites. In contrast, transcriptional activity induced
by either P419R or L422R mutants was completely absent in this context,
and E393R and L400R displayed 10 and 35% activation relative to the
wild-type TR, respectively. By contrast, the TR mutants T426R and
M430R, which impaired TR-RXR association in solution, were able to
activate transcription through GAL·RXR (Fig.
5). These data indicate that hydrophobic
residues at the center of the TR LBD dimer interface can abolish
transcriptional activities of TR linked to RXR LBD by presumably
preventing their interactions. The data also suggest that other
proteins in the cell stabilize the TR-RXR interactions for some TR
mutants relative to those observed under cell-free conditions.
In the current studies, a structurally guided scanning mutagenesis
of the TR and RXR LBD and DBD surfaces was used to infer the LBD
surfaces that form TR homodimers and TR-RXR heterodimers. This approach
has the advantage over studies using mutagenesis based on the linear
receptor sequence in that mutations placed on the outside surface of
the receptor are likely to disrupt local interactions with proteins and
much less likely to disrupt receptor folding, which complicates
interpretations of results. In support of this we found that the
mutations affecting dimerization and/or heterodimerization retained
other receptor functions such as ligand and coactivator binding. An
additional advantage of the surface scanning approach is that analyses
of multiple mutations demonstrate patterns and minimize erroneous
conclusions derived from interpretations of results from a single
mutant. These functional data can also determine whether the way
receptors crystallize with purified proteins reflects the functional
interactions in other conditions. Thus, we compared the interfaces
found in two TR LBDs that crystallized as dimers with those determined
from the functional studies. The data provide the first comprehensive
analysis directed at mapping the LBD interface for forming TR
homodimers and heterodimers with RXR. They suggest that a similar
surface participates in formation of TR homodimers with DR-4 and F2 DNA
and TR-RXR heterodimers in solution and with DR-4, F2, and TREpal.
The TR LBD crystals were obtained with both the rTR The scanning mutations were used first to define a TR-RXR LBD
heterodimer interface when the TRs are soluble and RXRs are linked to
GST (Fig. 2A). This method likely reflects interactions of
the two receptor types in solution. TR homodimer interactions were not
tested in this context, because they have not been observed in
solution. Mutations in the TR LBD that blocked GST·RXR-TR
interactions were located on a small surface comprising H10 and H11,
(Fig. 2, A and B), whereas 30 mutations on other
parts of the TR LBD surface failed to inhibit the reaction. Although
this heterodimer surface differed from that observed in our TR-TR LBD
homodimer crystals or of the PR homodimer (28), it resembles that
observed in the structures for the RXR Interestingly, the hydrophobicity of residues central to the TR
homodimer and TR-RXR heterodimer interactions, Leu422 and
Met423, is conserved in all the receptors, which exhibit
that interaction in the crystal (RXR Data in support of the notion that the TR utilizes the same LBD surface
for forming homodimers with DR-4 and F2 DNA as it uses for heterodimer
formation on GST is based on the observation that some of the same
mutations in this surface disrupted all of these interactions.
Variations in the effects of the mutations in the three contexts were
observed, and some of the mutations that affected the TR-RXR·GST
interaction failed to affect the homodimer interactions. However, the
overall pattern suggests at the least an overlapping surface, and the
same core of critical hydrophobic residues is employed by TR to form
homodimers and heterodimers with RXR. This pattern is similar to the
one found in the functional interface of growth hormone-growth hormone
receptor where a small and complementary set of contact hydrophobic
residues maintains binding affinity, a property that may be general to protein-protein interfaces (42). The surface centers on the Leu422 residue in TR H11, which disrupted TR homodimers on
DNA and TR heterodimers formed in solution. Whereas the overall surface
extends from residues Pro419 to Met430 in H11
and from Glu393 to Leu400 in H10 (Fig.
2B), the individual contribution of these residues varies
depending on the context in which the interactions take place. Thus,
for TR homodimers bound to F2, only Leu400 in H10 and
Leu422 and Met423 in H11 appear to be the
critical residues under the conditions studied, whereas flanking
residues such as Glu393 in H10 or Pro419 in H11
are critical for TR homodimer formation on DR-4. Finally, an even
larger set of flanking residues such as Thr426 and
Met430 are critical for TR-RXR heterodimers in solution.
That similar homodimerizing and heterodimerizing interfaces are used
for TR LBDs to bind to DR-4 and F2 elements provides support for the model that LBD dimer interfaces are independent of orientation of TRE
half-sites. Another interesting point derived from these studies is
that contrary to the prevailing model, the receptors do not need to
form dimers in solution before DNA binding. Thus, TR mutants incapable
of interacting with RXR in solution, such as E393R, L400R,
P419R, M423R, T426R, and M430R formed stable TR-RXR heterodimers with DNA.
Whereas mutations readily blocked TR binding to DR-4 and F2 elements,
no mutation, including L422R, disrupted homodimers on TREpal. These
data suggest that the "dimers" on TREpal consist of independent
binding of two monomers. This notion is supported by the observation
that "homodimers" on TREpal are formed very weakly as compared with
those on DR-4 and F2 (6, 7). Another indication that TR "dimers" on
TREpal are really two monomers is based on the fact that T3
increases TR binding as monomers with several TREs and as
"homodimers" on TREpal (6, 7), whereas T3 decreases TR
binding as homodimers with DR-4 or F2 elements (6, 7, 43, 44). The data
indicate that at least part of the TR surface that mediates both
dimerization on DR-4 and F2 and heterodimerization on GST·RXR also
mediates TR-RXR heterodimerization on DR-4, F2, and TREpal elements.
The first evidence supporting this conclusion is that L422R in the
center of the surface weakens formation of TR-RXR heterodimers on F2 (Fig. 3B, lane 16) and abolishes them on TREpal
(Fig. 3C, lane 16).
The finding that none of the TR LBD single mutants blocked TR
association with wild-type RXR with DR-4 suggested the possibility that
DNA may be stabilizing such interactions. This is supported by the
observation that heterodimerization on DR-4 was impaired when the TR
L422R mutant was incubated with the RXR DBD mutant. These findings
argue that interactions among TR and RXR DBDs play a role stabilizing
heterodimer formation on DR-4 and indicate a dominant role for the LBDs
relative to DBDs in stabilization of both homodimers and heterodimers
on DR-4. Our results further suggest that the analogous surface on the
RXR mediates the interaction of this receptor with the TR. Thus, a
double mutation in the RXR LBD, L419R/L420R greatly reduced formation
of heterodimers with wild-type TR on DR-4. An analogous double mutation
on the TR LBD, L422R/L423R, did not affect heterodimer formation with
DR-4 to the same extent (data not shown), suggesting that RXR has a
more dominant role in TR-RXR heterodimer formation and explain why, in
contrast to TR homodimers, TR-RXR heterodimers are formed in solution
and are not disrupted by ligand binding.
The formation of TR homodimers on DR-4 might be because of DBD-DBD
interactions, or might simply result from placement of the LBDs
together after independent binding of two receptor monomers to DNA. Our
findings that isolated TR LBD mutations inhibited TR homodimers, and
that TR DBD mutations did not, suggest that DBD-DBD interactions in TR
homodimers, if they exist, are not functionally important for processes
requiring dimers. These observations are consistent with results of
other investigators that failed to detect formation of TR DBD dimers on
DR-4 (20). If the DBDs do not interact in the TR homodimer, this could
explain the greater stability of the TR-RXR heterodimer on DR-4.
Interestingly, TR-RXR heterodimers are not stabilized by such DBD-DBD
interactions on F2, because, in contrast to what is seen on DR-4, the
weakened interaction between the TR L422R and wild-type RXR on F2 is
not further disrupted by mixing the TR L422R with the RXR DBD mutant (data not shown). However, TR-RXR heterodimers do form on TREpal, where
they appear to be weaker than the ones formed on F2 and DR-4, because
L422R disrupts them completely. We interpret that in the case of
TREpal, TR monomers recruit RXR using the same LBD dimer interface it
utilizes to bind RXR in DR-4 and F2, but that the resultant TR-RXR
complex bound to TREpal is less stable.
RXR serves as a heterodimer partner to VDR and RAR and it was predicted
that DBD-DBD contacts would stabilize VDR-RXR and RAR-RXR heterodimers
(23). However, we previously found that a single LBD mutant in the VDR
analogous to the L422R in the TR impaired the formation of heterodimers
with RXR on DR-3 (45) suggesting that DBD-DBD contacts do not
contribute to VDR-RXR heterodimers to the extent that they do with the
TR-RXR heterodimers. It is noteworthy in this respect that the DR-4
configuration places the center of the DBDs with a 10 bp separation,
which is approximately one turn of the DNA (10.5 bp) and aligns the
DBDs in a way that could optimize contacts between the DBDs. It is
possible that the shorter separation and 36° rotation expected
between the RXR and the VDR twists the orientation of the DBDs to
eliminate direct contact. In this context, VDR does not form homodimers
on DNA (45). This again points to properties of the RXR that are
dominant in forming the complex.
The current studies argue that TRs use a similar LBD surface for
homodimerization and heterodimerization. However, there may be
differences in the contacts made, even though they involve overlapping
surfaces. In this regard, it is known that proteins may possess
consensus sites with favorable intrinsic physiochemical properties that
are dominant for protein-protein interactions with multiple partners
(46). Modest adjustments occur within this flexible consensus site to
accommodate binding to different partners and the crucial properties
for this convergent binding surface are its accessibility,
hydrophobicity, and limited charge interactions at its periphery (46).
Such properties are indeed analogous to the ones we found in the dimer
surface of TR and RXR, where a patch of hydrophobic residues are
readily accessible for protein interactions. Crystal structures of
heterodimeric complexes of RAR We addressed whether the mutations could block functional
receptor-receptor interactions in vivo, by using a
heterologous system that employed the GAL-DBD linked to the RXR LBD and
TR mutants with a reporter gene containing GAL DNA binding sites. Several mutants that blocked formation of GST·RXR-TR heterodimers under cell-free conditions also blocked TR action with the heterologous system indicating that the preferred functional TR-RXR interface in vivo is similar to that found in vitro.
However, the mutants were overall less effective at disrupting these
in vivo interactions than those observed in cell-free
conditions, and therefore defined a more restricted area of required
tight contact. This could imply stabilization because of interactions
with endogenous factors, such as those recruited because of tethering
the TR and RXR in higher order complexes with coactivators and other
accessory proteins (47).
In summary, the data define a common domain of the TR and RXR that is
involved in TR-TR dimerization and TR-RXR heterodimerization. The
strength of the interaction varies and can be influenced by the
partner, DNA, DBD-DBD interactions and probably other proteins that
form higher order complexes in the cell. In some, but not all cases,
interactions between this domain on the TR are important for TR action.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (L365R and L372R) or human (h) TR
1 (L428R) disrupted
TR-RXR heterodimers, but not TR homodimers on TREpal (the unspaced
palindrome), DR-4 elements (direct repeat elements spaced by 4 base
pairs), and inverted palindromes (11, 17). Furthermore, single
mutations in arginine 316 to histidine, arginine 338 to tryptophan, and
arginine 429 to glutamine in hTR
1 disrupt homodimers but not
heterodimers (18). Whereas these data indicate differences in
requirements for heterodimerization and homodimerization, the
usefulness of these mutations for defining the TR homodimerization or
heterodimerization surfaces is limited, because these mutations are in
the interior core structure of the TR.
(24), ER
(25), ER
(26), PPAR
(27), and PR (28) LBDs
crystallized as homodimers. Crystals of LBD heterodimers were recently
reported for the RXR-RAR (29), where the interface is similar to that
observed in the RXR
, ER
, ER
, and PPAR
homodimers (24-27)
and for the PPAR
-RXR
heterodimer (30), where the interface is
slightly asymmetric with the PPAR LBD rotated ~10° relative to that
expected from the C2 symmetry axis of the RXR LBD. The residues in H10
and proximal region of H11 form more than 75% of the total LBD dimer
surface found in homodimers or heterodimers, whereas PR homodimers
contacts are in the distal part of H11 and H12. The PR dimerization
domain also covered a much smaller area (700 Å2) than that
for the ER
(1703 Å2), which led the authors to question
the physiological relevance of the observed dimer (31). In addition,
the structures of the RXR
LBD bound to 9-cis retinoic
acid (32) and of the PPAR
LBD bound to fatty acids (33) were solved
and showed two monomers per asymmetric unit. However, the packing
contacts found between these monomers did not resemble those present in
the unliganded RXR
LBD or in the PPAR
LBD homodimers suggesting
that a different dimeric conformation may be found with these
receptors. Altogether, these structural data indicate that in the
absence of DNA, nuclear receptors form dimers by engaging H11 and
possibly other helices in diverse macromolecular interactions that may
give rise to varied dimeric conformations.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 sequence. Some mutations were created using QuickChange
site-directed mutagenesis kits (Stratagene). The mutated sequences were
verified by DNA sequencing using Sequenase Kits (Stratagene). These TR
mutants changed the amino acids in the hTR
1 surface that were
selected for scanning mutagenesis using the x-ray structure of the
TR
LBD as a guide to localize the surface residues on hTR
1 (16,
34). Subsequent solution of the hTR
LBD structure (35) revealed that
the mutations were placed correctly. Most mutations changed the
resident amino acid to arginine, reasoning that bulky and charged
residues on the surface were more likely to disrupt TR association with
other proteins over a wider range without reducing TR solubility. Each mutant maintained the overall integrity of TR as judged by the ability
to bind [125I]T3 with Kd
values between 4 and 174% of that for the native receptor and/or to
bind the glucocorticoid receptor interacting protein 1 (GRIP-1) fused
to GST as effectively as the wild-type TR. We initially tested, as
reported previously, widely separated locations to probe the entire LBD
surface for dimerization (34). Subsequently, a series of mutations were
performed along H10 and H11 in a region where residues were identified
that disrupted dimerization to define better the structural
requirements for dimerization. Mutations are represented by the
single-letter amino acid abbreviation of the native residue followed by
its position number in the sequence and the mutated residue
abbreviation. The affinity of the wild-type TR
1 is 60 ± 7 pM.
--
Crystallization conditions for hTR
LBD with Triac
has been described fully (35). Briefly, crystals are obtained by
hanging drop vapor diffusion (12 h, 25 °C) from a drop (1 µl of
TR
LBD solution at 9 mg/ml, 1 µl precipitant solution) suspended
above a reservoir composed of 1.0 M sodium acetate
[NaH3OAc] and 0.1 M sodium cacodylate
[NaCac], pH 7.2. Crystals were of space group P3121
(a = 68.9 Å, c = 131.5 Å) and
contained 1 molecule of TR
LBD. The N-terminal His tag was not
removed prior to crystallization.
--
Hexagonal crystals of rTR
LBD with T3
were obtained using hanging drop vapor diffusion at 17 °C from a
drop containing a 1:1 mixture of ~10 mg/ml protein, 3 mM
dithiothreitol with well solution containing 0.45 M
(NH4)H2PO4 and 0.2 M
(NH4)2SO4 buffered with 0.1 M KH2PO4, pH 7.0. The space group
was determined to be hexagonal, P61(5)22 from oscillation
data; the correct enantiomorph P6522 was identified through
molecular replacement using the model of the rTR
LBD (16). The
crystals contain one monomer/asymmetric unit. The unit cell dimensions
of these crystals are a = b = 108.64 Å, c = 133.72 Å.
--
Structural analysis of hTR
LBD with Triac has
been recently described fully (35). Briefly, crystals were transferred
into a cryoprotectant composed of 30% glycerol, 1.4 M
NaAc, and 100 mM NaCac, pH 7.2, then suspended in a nylon
loop attached to a mounting pin, and flash frozen (liquid nitrogen).
Diffraction data were measured to 2.4 Å using synchrotron radiation at
Stanford Synchrotron Radiation Laboratory beamline 7-1 (
l = 1.08Å) and processed using HKL. A molecular replacement solution was
found using AMORE from the model of the rTR
LBD, with ligand omitted (36). Refinement with CNS produced a model with an
Rcryst of 24% and an
Rfree of 26%.
--
A cryoprotectant composed of 30% glycerol, 0.5 M (NH4)H2PO4, 0.2 M (NH4)2SO4, and 0.1 M KH2PO4, pH 7.0 was used, and a
complete data set measured at SSRL (
l = 1.08Å) and processed
using HKL. A molecular replacement solution for the Form II crystal was
found using AMORE from the rTR
LBD model with ligand removed (16). The resulting electron density maps show interpretable density for the
T3 ligand; furthermore, an anomalous Fourier map calculated from the molecular replacement phases identifies the sites for the
iodine scatterers consistent with the ligand density. Refinement with
CNS produced a model with an Rcryst of 25% and
an Rfree of 28%.
was prepared in
Escherichia coli strain HB101 as a fusion protein with
glutathione S-transferase (GST) as per the manufacturer's
protocol (Amersham Pharmacia Biotech). The binding experiments were
performed by mixing glutathione-linked-Sepharose beads containing 10 µg of GST·hRXR
fusion proteins (Coomassie Plus protein assay
reagent, Pierce) with 1-2 µl of the 35S-labeled
wild-type or mutant hTR
1 (30 fmols) in 150 µl of binding buffer
(20 mM HEPES, 150 mM KCl, 25 mM
MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitors)
containing 2 µg/ml bovine serum albumin for 1.5 h. Beads were
washed three times with 1 ml of binding buffer, and the bound proteins
were separated using 10% SDS-polyacrylamide gel electrophoresis and
visualized by autoradiography.
1 mutants to DNA was assayed by gel
shift assays by mixing 30 fmols of radiolabeled TRs
([35S]Met, DuPont) produced in a reticulocyte lysate
system, TNT® T7 Quick (Promega), with ~600 fmols (10 ng) of
nonradiolabeled DR-4 or F2 oligonucleotides or 120 fmols (2 ng) of the
TREpal oligonucleotide and 2 µg of poly(dI-dC) (Amersham Pharmacia
Biotech) in a 20-µl volume reaction as previously described (7). The binding buffer contained 10 mM NaPO4, 1 mM MgCl2, 0.5 mM EDTA, 20 mM NaCl, 5% glycerol, 0.1% monothioglycerol. After 20 min
at room temperature, the mixture was loaded onto a 5% nondenaturing polyacrylamide gel that was previously run for 30 min at 200 V. To
separate the TR·DNA complexes, the gel was run at 4 °C for 150-240 min at 240 V, using a running buffer containing 6.7 mM Tris-base (pH 7.5 for a 10× stock at room temperature),
1 mM EDTA, and 3.3 mM sodium acetate.
1 expression vector was cotransfected with the 2 µg of the GAL·RXR (GAL4 DBD fused to hRXR
LBD) expression
vector. The reporter gene contained five GAL binding sites upstream of the adenovirus E1b minimal promoter linked to luciferase coding sequences (LUC). The LUC activity was analyzed after the cells were
incubated 18-24 h with the hormone (Luciferase Assay System, Promega).
:mRXR
heterodimer (Ref. 29, Protein Data Bank
(PDB) entry 1DKF). To create the model of the TR-RXR heterodimer, the
rTR LBD was superimposed on hRAR using the C
atoms of the
hydrophobic core helices H3-H5-H6 (rTR residues
Ile226-Arg266; hRAR residues
Ile236-Arg276), and H9 (rTR residues
Asp313-Leu322; hRAR residues
Asp323-Met332), using LSQMAN (r.m.s. deviation
of 0.55 Å for 51 C
; r.m.s. deviation of 1.42 Å for 203 C
after
optimization). Hydrophobic and hydrophilic surface buried in the
crystallographic contacts was calculated using ASC, with a 1.4 Å probe
(38, 39). Hydrogen bonds were identified using HBPlus (40).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and TR
subtypes exhibit two distinct symmetrical intermolecular contacts. In
TR
, the individual monomers are rotated 180° with respect to each other, with an axis of symmetry parallel to H11, such that the helices
cross with the junction near the mid-point of H11 (Fig. 1A). The contact buries a
modest 550 Å2 of surface area per monomer. The primary
contacts are between residues in H11, with contributions from the
C-terminal portion of H8. A cluster of hydrophobic residues in H11
marks the center of symmetry: Met376, Ile377,
Ala378, and Cys379. At the periphery of the
contact are electrostatic interactions between residues in H8 and H11:
Lys288 with Asp326 and Glu301 with
Asp373.
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Fig. 1.
Ribbon drawings of TR LBD
homodimers. A,
rTR 2 and B, hTR
(35). To
facilitate comparison of the homodimers, the left monomer in each
appears in identical orientation (i.e. viewed down H10). The
helices discussed in the text are labeled: H8 (blue-green),
H10 (yellow), H11 (dark blue), and H12
(magenta).
, the individual monomers are again rotated 180° with respect
to each other, but the axis of symmetry is perpendicular to H11,
producing an antiparallel LBD dimer (Fig. 1B, Ref. 35). Rather than crossing as in the TR
, H11 of one monomer nestles between H8 and H11 of the other monomer. The contact buries 580 Å2 of surface area per monomer. The residues in the
contact again involve H11 but with the opposite end of H8. Hydrophobic
contacts in H8 pack Val348 against Val348 and
Ala352 in the other monomer. Hydrophobic contacts in H11
are made by Met423 and the aliphatic stem of
His441 and between the hydrophobic cluster
Met430, Ala433, and Cys434, just as
in the TR
. Electrostatic interactions link Arg338 from
strand S4, Lys342 in H7, and Asp355 in H8; and
also Asp427 and His441 in H11, with an
additional hydrogen bond between Asp427 and
Ser437.
1 bound to GST·RXR in a T3-independent manner (Fig. 2A), whereas binding of
wild-type or mutant TRs to GST alone was negligible
(data not shown). Replacing amino acids in H10 (Glu393 and
Leu400) and H11 (Pro419, Leu422,
Met423, Thr426, and Met430)
with Arg greatly diminished TR interactions with RXR (Fig.
2A). This was not observed with the mutants E390R in H10 and
H416R and C434R in H11 (Fig. 2A) or 62 other mutants
described under "Materials and Methods" and spanning helices 1, 3, 5, 8, 9, and 12 (data not shown). Fig. 2B shows the location
of the amino acids involved for TR association with RXR in solution,
from the GST·RXR interactions with TR mutants. All of the TR mutants
defective in forming heterodimers with GST·RXR bound T3
and interacted with GST·GRIP1 as well as the wild-type TR (data not
shown). Taken together, these results define a relatively small surface
in H10 and in the amino-terminal portion of H11 of the TR LBD that is necessary for binding RXR in solution.
View larger version (40K):
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Fig. 2.
RXR binding by hTR 1
wild type and mutants. A, in vitro binding of
35S-labeled hTR
1 wild type and mutants to GST·hRXR
(18). The binding assay was analyzed by autoradiography after
separation using 10% SDS-polyacrylamide gel electrophoresis. The
mutations with their helix locations are indicated above the gels. On
the top we indicate the Input (40% of the amount
used in the reaction) of each TR and on the bottom we show
the TR binding to GST·RXR
. B, scheme showing the hTR
LBD structure with
-helices drawn as ribbons and
-strands as arrows depicting the residues
Glu393 and Leu400 in H10 (green) and
Pro419, Leu422, Met423,
Thr426, and Met430 in H11 (dark
blue) inferred by mutations to form heterodimers with RXR in
solution.
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Fig. 3.
Formation of homodimers or heterodimers of
wild-type hTR 1 or mutants on diverse
TREs. Gel shift assays contained 40 fmols of the in
vitro-translated 35S-labeled TRs (usually 1-3 µl),
3 µl of either unprogrammed lysate or unlabeled hRXR
and 10 ng of
DR-4 (A) or F2 (B) or 2 ng of TREpal
(C). The mutations with their putative helix locations are
indicated above the gels. D, the surface of the hTR
LBD
is shown indicating H10 and H11 and the residues proposed to contribute
to the dimer surface. Areas in the helices are colored according to
their importance for dimerization. The surface represented in
red forms an area surrounded by the hydrophobic amino acids
Leu400, Pro419, Leu422, and
Met423; the surfaces in green and
blue represent adjacent residues that are progressively less
important for formation of TR homodimers on DR-4 and F2.
:mRXR
heterodimer (Ref. 29, PDB entry 1DKF) allowed us to propose a model for
the TR-RXR heterodimer, which is shown in Fig. 4B, depicting
the major interactions between the hydrophobic residues in H10 and H11
of TR with H11 of RXR.
View larger version (55K):
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Fig. 4.
Characterization of heterodimer formation
between TR and RXR on DR-4. A, gel shift assays
contained 40 fmols (usually 1-3 µl) of the in vitro
translated 35S-labeled wild-type hTR 1 or LBD (L422R) or
DBD (D177A) mutants mixed respectively with 3 µl of either unlabeled
unprogrammed lysate (lanes 1, 5, 9) or with
hRXR
wild type (lanes 2, 6, 10), hRXR
DBD
mutant (lanes 3, 7, 11), or hRXR
LBD mutant L419R/L420R
(lanes 4, 8, 12). B, model for the TR
1-RXR
LBD heterodimer interface. The inset highlights the
interactions between the hydrophobic residues Leu400 in H10
(green) of TR with Leu420 of H11 (dark
blue) of RXR and Leu422 and Met423 in H11
of TR with Leu419 in H11 of RXR.
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Fig. 5.
Specific effects of mutations on
hTR 1 transcriptional activation in CV-1
cells. Cells were cotransfected with 1 µg each of expression
vectors encoding a GAL·RXR LBD fusion protein, and wild-type or
mutant hTR
1, and 5 µg of a reporter gene containing 5 GAL binding
sites and encoding luciferase. After culturing with or without 100 nM T3, cell extracts were assayed for
luciferase activity. T3 activation was expressed as the
percentage of wild-type TR. The wild-type or different hTR
1 mutants
are indicated. The data show a representative experiment, which was
repeated 3-5 times for each TR mutant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and hTR
LBDs.
In both structures, the TR monomers were rotated 180° with respect to
each other. With the rTR
, the helices crossed with a junction near
the middle of H11, whereas with the hTR
, the axis of symmetry is
perpendicular to H11, producing an antiparallel LBD dimer in which one
monomer lies between the end of H8 (opposite to that with the rTR
)
and H11 of the other monomer. The contacts bury a modest 550 Å2 and 580 Å2 of surface area per monomer for
the rTR
and hTR
LBDs, respectively. These observations could
imply a weak interaction because the bona fide
protein-protein interface typically buries > 700 Å2
of solvent-exposed area (41). In support of this notion, none of the
mutations of residues of either H8 or H11 involved in these contacts,
such as Asp355, Ala433, Cys434, and
Ser437, disrupted TR-TR dimer formation on DNA, whereas
mutations of other residues of H10 and H11 did affect these
interactions. These observations, taken together, suggest that the
contacts observed in the TR crystal structures are not involved in
formation of TR-TR homodimers on DNA and also differ from the contacts
reported in the structures of RXR and PR homodimers (24, 28).
(24), the ER
(25), the
ER
(26), and the PPAR
(27) homodimers. In other studies the functional protein-protein interfaces derived from mutational data have
been found to differ from those that form in the crystal. For example,
about 30 side chains from growth hormone and its receptors make
contact, but individual replacement of contact residues with alanine
showed that only a central hydrophobic region accounts for more than
three-quarters of the binding free energy (42). Furthermore, mutations
in interface residues cause structural changes that redistribute the
particular contributions of individual residues to the free energy of binding.
(24), ER
(25), ER
(26),
PPAR
(27)). By contrast, in the PR and the other steroid receptors
with the exception of ER, these residues are hydrophilic or charged,
resembling the mutations introduced in the TR that disrupt the
interaction. Thus, the PR homodimer interaction could be representative
of one class of LBD interactions, the classic "homodimer"
receptors, whereas the interactions examined here are representative of
the "heterodimer" receptors.
and RXR
LBDs (29) and PPAR
and
RXR
LBDs (30) revealed that the helices of these receptors, which
correspond to H10 and H11 of TR, contribute more than 75% of the dimer
surface. A hydrophobic core of residues analogous to the ones described here for the TR-RXR LBD interactions is at their centers. Our model for
the heterodimeric interface of TR and RXR LBDs based on functional
studies closely matches the surfaces reported for the crystal
structures of RAR
-RXR
and PPAR
-RXR
LBD heterodimers. This
finding supports the contention that the conservation of interface
residues of this subgroup of nuclear receptors, which is significantly
higher than that of the entire LBD, enables these receptors to engage
in heterodimer formation with RXR (29) and predicts that the LBD
surfaces for heterodimerization are likely to be similar within this
subgroup of nuclear receptors.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Brian West for providing TR mutants used in this study and Dale Leitman and Francisco Neves for reviewing the manuscript. Some of the molecular graphics work was performed using the Computer Graphics Laboratory at the University of California, San Francisco (NIH P41-RR01081).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK41842 (to R. J. K.) and DK09516 (to J. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a fellowship from the Brazilian Research Council (CNPq). To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Faculty of Health Sciences, University of Brasília, UnB, Brasilia, DF 70910-900 Brazil. Tel.: 55-61-307-2098; Fax: 55-61-347-4622; E-mail: ralff@unb.br.
Recipient of a postdoctoral fellowship from the National
Institutes of Health.
Proprietary interests in and serves as a consultant and Deputy
Director to Karo Bio AB, which has commercial interests in this area of research.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M010195200
2 R. L. Wagner, B. R. Huber, J. W. Apriletti, B. W. West, J. D. Baxter, R. J. Fletterick, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: TR, thyroid hormone receptor; LBD, ligand binding domain; DBD, DNA binding domain; RXR, retinoid X receptor; RAR, retinoic acid receptor; ER, estrogen receptor; VDR, vitamin D receptor; PR, progesterone receptor; PPAR, peroxisome proliferator-activated receptor; GRIP-1, glucocorticoid receptor interacting protein 1; T3, 3,5,3'-triiodo-L-thyronine; TRE, thyroid hormone response element; DR, direct repeat; DR-4, direct repeats spaced by 4 base pairs; F2, inverted repeats with a 6-base pair separation; TREpal, inverted repeats without a nucleotide separation; GST, glutathione S-transferase; r.m.s., root mean squared.
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