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INTRODUCTION |
Steroid hormones regulate the transcription of target genes in the
cell by binding to transcription regulators that belong to the
superfamily of nuclear receptors. All members of this family display a modular structure composed of six domains (A-F). The E
region constitutes the ligand-binding domain
(LBD)1 containing a
ligand-dependant transactivation function (AF-2) (1, 2). The
transcriptional activity of nuclear receptors is mediated by
interactions with the transcriptional machinery through various
corepressors and coactivators (3). Their ability to modulate gene
expression in a ligand-regulated manner is based on the position of
helix H12 carrying the AF2-AD transactivation function (4). Several
positions of H12 have been observed (5). In the absence of ligand, H12
has been shown to be exposed to solvent (6). Ligand binding triggers a
conformational change that results in the repositioning of H12 on the
core of the LBD, closing the ligand binding pocket like a lid (7). This
is referred to as the mouse trap mechanism (8). In agonist-bound LBDs a surface suitable for coactivator binding is then created (9-12). In
most antagonist-bound complexes (11, 12), H12 has been observed
positioned in a structurally conserved cleft where the LXXLL motif of
the coactivator molecule binds. These observations suggest a mechanism
for antagonism where H12 and the coactivator compete for a common
binding site. Note that the agonist position of H12 is unique, whereas
its position in antagonist-bound complexes is not. Therefore knowledge
of the features responsible for inducing and stabilizing a given
conformation is a key step in understanding the initial events of
nuclear receptor transactivation.
Several crystal structures of both ER isotypes (ER
and ER
) have
been solved in complex with natural and synthetic ligands (12-16). The
natural ligand 17
-estradiol acts as a pure agonist on both isotypes.
Others typified by EM-800 and ICI164,384 are described as pure
antagonists (17). A third category of ligands displaying cell-type and
promoter dependence in ER regulation are referred to as selective ER
modulators (SERMs) (18). SERMs such as raloxifen and
4-hydroxytamoxifen efficiently antagonize the AF2, but not the AF1
function, and act as a pure antagonist (19) in ER
, which seems to
lack a functional AF1 domain (20). The features responsible for
inducing a given conformation and stabilizing it are crucial to the
definition of the optimal stereochemical and biophysical specificity of
a ligand. Here we present the comparison of the wild type hER
LBD
crystal structure (16) with that of a mutant protein complexed with
estradiol, where three cysteine residues were mutated in serine. The
mutant protein binds estradiol with wild type affinity but has limited
transcriptional capacity. In the structure of the Cys
Ser
triple mutant hER
LBD, we observed an antagonist conformation
despite the presence of a tightly bound estradiol in the ligand-binding
cavity. This antagonist conformation, together with the transcriptional
activity of the single, double, and triple cysteine to serine mutant
receptors, supports the view of the agonist-antagonist equilibrium of
H12 and gives some insight into the molecular mechanism for the
conformational switch that drives the receptor in an agonist or
antagonist conformation.
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EXPERIMENTAL PROCEDURES |
Protein Production, Purification, and Crystallization--
The
Cys
Ser triple mutant hER
LBD (Lys302
Pro552), in fusion with six histidine residues is produced
using the pET15b/Escherichia coli BL21(DE3) expression
system and purified by a zinc affinity column, ion exchange, and gel
filtration. The purification procedure is similar to that of the wild
type ER LBD (16). Crystals were obtained by vapor diffusion at 4 and
17 °C using hanging drops made by mixing 1 µl of protein solution
(2.5 mg/ml) with 1 µl of reservoir solution (12% polyethylene glycol
8000, 0.4 M NaCl, 100 mM imidazole, pH = 6.9). Prior to data collection, crystals were flash-cooled in liquid
ethane after a fast soaking in a cryoprotectant buffer (20% glycerol,
15% polyethylene glycol 8000, 0.4 M NaCl, 100 mM imidazole, pH = 6.9). Crystals belong to the space
group P6522 with cell parameters a = b = 58.6 Å, c = 276.02 Å,
=
= 90°,
= 120° (one monomer in the asymmetric
unit, 45% solvent).
Structure Determination--
X-ray data were collected at 120 K
in a nitrogen gas stream using synchrotron radiation (European
Synchrotron Radiation Facility Laboratoire pour l'Utilisation du
Rayonnement Éléctromagnétique). The
diffracted intensities were processed using the programs DENZO and
SCALEPACK (21). Experimental phases were obtained using gold and
platinum derivatives. The multiple isomorphous replacement analysis was performed using CCP4 (22) and SHARP (23) packages. It
enabled the construction of the complete model. An initial map was
calculated to 3-Å resolution using multiple isomorphous replacement phases and solvent flattening using SOLOMON (22, 23). Refinement was performed with CNS (24) using bulk solvent corrections. All data between 15- and 2.2-Å resolution were included with no sigma cutoffs (Table
I).
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Table I
Data processing, phase determination and refinement statistics of Cys
Ser triple mutant structure (P6522, a = b = 58.6 Å, c = 276.0 Å)
Rsym = h i|I(h) I(h)i |/ h i
I(h)i, where
I(h)i is the average intensity of
reflection h, h is the sum of the measurements of
reflection h. Rcullis = lack of
closure/isomorphous difference. Phasing power = Fh/lack of closure. Rmsd, root mean square
deviation. fom, figure of merit.
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The dimer interface was calculated with the Grasp package (25). The
buried interface, calculated with non-hydrogen atoms only, was obtained
with the excluded area method, which calculates the accessible surface
regions of protomer A buried by protomer B.
ER Expression Vectors and Reporter Plasmids--
pSG5-HEGO (26),
pG4M polyII-ER(DEF) (27) for full-length ER and GAL-ER
eucaryotic expression, respectively, are used in transactivation
assays. Vit-tk-CAT (28) for full-length ER transcriptional activity and
17m-tk-CAT (29) for GAL-ER activity are used as reporter plasmids.
CMV-
Gal served as an internal control to normalize for transfection
efficiency. Bluescript KS+ plasmid was used as carrier DNA.
The procaryotic expression system pET15b-LBD/BL21(DE3) (Novagen) was
used for estradiol binding ability and structural studies of the
LBD.
Mutagenesis and Cloning--
The mutations were generated in
different constructs to allow structural and functional studies.
Cysteine to serine mutations at positions 381, 417, and 530 were
introduced in the LBD cloned in NdeI-BamHI sites
of pET15b by PCR-assisted mutagenesis, using Deep Vent DNA polymerase
(Biolabs) and the appropriate oligonucleotides. Triple mutant
Cys
Ser His-tagged LBD was used in the structural studies
and ligand binding assays. For transactivation assays the single,
double, or triple mutants, in all possible combinations, were brought
into the full-length receptor (pSG5-ER26) by digesting the LBD with the
restriction enzymes HindIII-BglII. This fragment of 252 base pairs sharing the mutation C381S and/or C417S was inserted
in the HindIII-BglII sites of pSG5-ER. The
presence of another BglII cleavage site in pSG5 causes the
loss of a fragment of 547 base pairs. This
BglII-BglII fragment with or without the C530S
mutation was reinserted in the vector. The
BglII-BglII C530S fragment was obtained by
PCR-assisted site-directed mutagenesis. To remove the AF1 contribution,
the triple mutant was also brought into the
XhoI-BamHI sites of the pG4M vector
by PCR cloning using the pSG5-ER triple mutant as template, leading to
the eucaryotic expression of GAL-ER(DEF).
Based on structural observations, another triple mutant was designed
(E339A,E419A,K531A). These mutations were successively generated by PCR
mutagenesis in the DEF region (282) subcloned in the
XhoI-BamHI sites of pG4M. These
mutations were also brought in the LBD subcloned in the
NdeI-BamHI sites of pET 15b. All constructs were
verified by automated DNA sequencing.
Cell Culture and Transfection--
COS1 cells, an estrogen
receptor-deficient cell line, were transferred in phenol red-free
Dulbecco's modified Eagle's medium supplemented with 5%
charcoal-dextran-treated fetal calf serum and antibiotics (40 µg/ml
gentamicin, 0.1 mg/ml streptomycin, 500 units/ml specillin). Cells were
plated in six-well dishes (Costar) at a density of ~5 × 105 cells/well in a humidified 5% CO2
atmosphere at 37 °C. The cells were transfected 5-6 h later by the
calcium phosphate coprecipitation technique with 0.2 µg of wild type
or mutant receptor plasmid, 2 µg of reporter plasmid, 0.5 µg of
CMV-
Gal (internal control plasmid), and 7.3 µg of Bluescript
KS+ carrier DNA. These plasmids were mixed in 420 µl of
10 mM Tris-HCl, pH 8, 0.05 mM EDTA. 60 µl of 2 M CaCl2 was added by dripping. The mixture was dripped in 2× 480 µl of HBS (280 mM
NaCl, 50 mM HEPES, 1.5 mM
Na2HPO4·12H2O, pH 7.12)
and left 30 min at room temperature before being dispersed on the
cells. 300 µl were taken per well. The precipitate remained in
contact with the cells for 15 h. After this exposure, the cells
were washed with phenol red-free Dulbecco's modified Eagle's medium
and antibiotics. Cells were then incubated in culture medium containing
the indicated concentrations of estradiol for 24 h. Lysis was
achieved in 300 µl/well in 10 mM MOPS, 10 mM
NaCl, 1 mM EGTA, 1% Triton X-100, pH 6.5, for 30 min at
room temperature. The cellular lysates were centrifuged for 10 min at
16,000 × g.
CAT Assays--
The CAT was quantified by enzyme-linked
immunosorbent assay (Roche Molecular Biochemicals) according to the
manufacturer's recommendations. The amount of CAT was standardized for
transfection efficiency with the
-galactosidase activity in each
lysate. The basal level was defined as the CAT activity in cells
transfected with the reporter plasmid in the absence of receptor
plasmid. Each experiment was performed at least three times in
duplicate. Results are expressed in relative CAT activity in percent of
maximal wild type receptor activity (Fig. 3).
Estradiol Binding Ability of the LBD--
We have used the
pET15b-LBD/BL21(DE3) system to produce hER
LBD for ligand binding
assays. E. coli BL21(DE3) expressing native or mutant LBD
were lysed by sonification in the binding buffer (1 M
SB201, 50 mM NaCl, 50 mM Tris-HCl, pH 8, 1 mM EDTA, 1 mM dithiothreitol). After
centrifugation at 14,000 × g during 1 h at
4 °C, the soluble fraction (crude extract) was used for receptor quantification and dissociation constant (Kd)
determination. The total amount of protein in the crude extract was
quantified using the Bradford technique. The binding assays were all
performed in the presence of a total protein concentration of 5 mg/ml
to avoid retention of the receptor in complex with the ligand, thus the
crude extract was diluted with soluble proteins from untransformed bacteria. Receptor quantification was achieved in the presence of
10
8 M
[6,7-3H]estradiol (E. I. du Pont de Nemours & Co.E. I.) with (for nonspecific binding) or without (for
total binding) an excess of cold estradiol (2.10
6 M) and increasing amounts
of crude extract. After 5 h at 4 °C, bound (B) and free (F)
ligands were separated by dextran-coated charcoal (4% Norit A
charcoal, 0.4% dextran T-70 in the binding buffer). This mixture was
left on ice for 5 min and centrifuged at 12,000 × g
for 5 min. The supernatant was removed for scintillation counting (30).
Specific binding was plotted against the volume of crude extract for
receptor quantification. For the Kd determination
the crude extract was incubated with increasing concentrations (from
10
10 to 10
7
M) of radiolabeled estradiol at 4 °C overnight. Each
measure was done in triplicate for Scatchard analysis. The variation of B/F as a function of B was analyzed as described previously
(31).
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RESULTS |
The Molecular Structure--
The structure of the triple mutant
ER
LBD (Fig. 1b) exhibits
the predominantly
-helical fold observed for all nuclear receptors. The superposition over the wild type structure in complex with estradiol (16) (Fig. 1a) leads to an r.m.s. deviation of
0.54 Å over 211 C
atoms (H1, H3-H8, H9-H11). The most striking
conformational difference between these two structures is the different
positioning of helix H12 and the concomitant shortening of helices H3
and H11 (Fig. 1, a and b). In the mutant, the
activation helix is in the antagonist position as observed in the
raloxifen and tamoxifen complexes (12, 13). Both antagonist
structures superimpose very well to that of the mutant LBD. The r.m.s.
deviation is 0.5 Å over 213 C
atoms (H1, H3-H8, H9-H11, H12) and
0.5 Å over 238 C
atoms (H1-H6, H7-H11, H12) for the
raloxifen and tamoxifen complexes, respectively (Fig.
1c). The loop L8-9 is not seen in raloxifen, and the
structure of the loop L1-3 is closer to the triple mutant in the
complex with tamoxifen than that with raloxifen. Wild type and
mutant homodimers can be superimposed with a r.m.s. deviation value of
0.64 Å over 418 C
atoms. The helices H9 and H10 form the core of
the interface and contribute to more than 70% of it. Despite this good
match, the contributions of the secondary structure elements to the
interface, spanning the helices H7 to H11, differ among the two forms.
Due to the antagonist conformation of the mutant structure, helices H7,
H9, and H10 exhibit a smaller contact surface area (1475 Å2) compared with the wild type (1686 Å2).

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Fig. 1.
a, schematic drawing of a hER
wild type LBD with the bound estradiol. The cysteine residues are
depicted as yellow spheres. The AF2-AD containing helix H12
(red) is in the agonist position. b, the
C381S,C417S,C530S hER triple mutant LBD complexed to estradiol. H12
(red) is in the antagonist position. The now serine residues
are depicted as pink spheres. c, superposition of
wild type, Cys Ser triple mutant and the complex with tamoxifen
(12). Regions that superimpose perfectly are represented in
gray, whereas regions that differ among the structures are
colored in yellow for wild type LBD, pink for Cys
Ser triple mutant, and cyan for the tamoxifen
complex.
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The Ligand Binding Pocket--
The overall structure of the pocket
is similar in the wild type and the Cys
Ser triple mutant. All side
chains of the hydrophobic residues lining the pocket are at the same
position. This explains the fact that the dissociation constants at
equilibrium (Kd) between the wild type and
the Cys
Ser triple mutant for
estradiol are very close (Table II and Fig.
2). The main differences are found on the
17-OH and 3-OH side of estradiol (Fig. 3). Due to the antagonist
position of H12, the cavity in the triple mutant is not sealed as in
the wild type structure. On the O17 side (D-ring side) of
estradiol, the cavity reaches the surface of the protein and results in
a much larger volume than the wild type ligand binding pocket. This
channel is partially filled with water molecules, forming numerous
hydrogen bonds with the protein. On the 3-OH side (A-ring side) an open
narrow tunnel filled with water molecules is present in the mutant. In
the wild type, this tunnel is almost closed and only the water molecule
interacting with the 3-OH of estradiol is present. In the Cys
Ser
triple mutant the estradiol A-ring superimposes perfectly with its
equivalent group in the wild type complex, whereas the
D-ring is slightly shifted, as shown by the displacement of
the C17, which moves 0.5 Å closer to helices H3 and H12 (Fig. 3).
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Table II
Affinity constant (KD in nM), estradiol efficient
concentration (EC50 in nM), and
raloxifene-inhibiting concentration (IC50 in nM)
are compared for wild type and mutant GAL-ERs
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Fig. 2.
a, estradiol binding ability of wild
type and mutant ER ligand-binding domains. The graph represents typical
Scatchard plots for each LBD. Wild type ( ), C381S+C417S+C530S triple
mutant ( ), and E339A+E419A+K531A triple mutant ( ) LBDs are shown.
b, comparison of dose-response curves of transactivation
(expressed in relative CAT activity as a function of estradiol
concentration) for wild type GAL-ER LBD ( ), GAL-ER C381S+C417S+C530S
triple mutant ( ), and GAL-ER E339A+E419A+K531A triple mutant ( )
LBDs, showing that the ligand-independent transactivation function AF1
is not involved in the transcriptional activity of mutant receptors.
c, relative CAT activity (percent) in function of
raloxifen concentration (nM) at a constant
concentration of estradiol (10 nM) for wild type GAL-ER LBD
( ), GAL-ER C381S+C417S+C530S triple mutant ( ), and GAL-ER
E339A+E419A+K531A triple mutant ( ) LBDs. Anti-estrogens inhibit
estradiol-induced transcriptional activity of wild type and mutant
GAL-ER.
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The superposition of the Cys
Ser triple mutant with the
hER
-raloxifen complex reveals that Asp351, which
anchors the ammonium moiety of raloxifen, adopts the same conformation in both structures. All the helices, including H12, match
perfectly (r.m.s. deviation: 0.5 Å), the protruding chain of
raloxifen fitting perfectly in the water channel observed in the mutant structure. Furthermore, in this structure, electron density
could be observed for the loop 11-12, a region that was not seen in
the ER LBD/raloxifen structure. This loop includes the C
terminus of the shortened helix H11, in particular Lys529,
which points toward the ligand binding pocket channel.
ER pure antagonists exhibit acidic moieties in their protruding chain
and are thus unlikely to interact with Asp351 as do
raloxifen and tamoxifen. The present structure suggests Lys529 as a potential hydrogen bond partner for pure
antagonist ligands bearing sulfinyl-like (ICI182780) (32) or
sulfonyl-like (RU58668) (33) groups in their protruding chain. Such a
contact would cross the AF2 AD groove and hamper the agonist
positioning of H12.
Cumulative Effect of Cys
Ser Mutations on ER Transactivation
Potency--
Interestingly each single Cys
Ser mutation
(C381S,C417S; C530S) contributes equally to the observed
transactivation reduction for the triple mutant receptor (Fig.
4 and Table II). Each single Cys
Ser
mutation decreases the full-length receptor's activity by about 20%,
whereas double mutations reduce CAT activity by ~40%, and the triple
mutant exhibits a 56% decrease. Maximal wild type activity could not
be restored even in the presence of saturating estradiol
concentrations. Moreover each time a cysteine is mutated to serine the
ligand dose-response curve of the mutant receptor is slightly shifted
to the right, leading to a 6-fold shift in the ligand-efficient
concentration to trigger half-maximal activity (EC50 = 4.0 ± 1.5 nM) for the triple mutant, compared with
the wild type receptor (EC50 = 0.7 ± 0.1 nM, Table II). To investigate the contribution of the AF1
on transcriptional activity, we used the chimeric receptors (GAL-ER)
wild type and triple mutant on which the contribution of the
ligand-independent transactivation function AF1 is removed. The Cys
Ser triple mutant displays 48% activity compared with the wild type
GAL-ER, which is very close to the value observed for the full-length
triple mutant receptor (44%, Fig. 2b). These data showed
that the residual transactivation activity in the triple mutant is not
due to AF1. Antagonist competition assays with raloxifen
revealed that this SERM represses more efficiently the
estradiol-stimulated CAT activity of the Cys
Ser triple mutant
GAL-ER than that of the wild type (Fig. 2c). These data suggest that the activation helix can be more easily displaced from
its optimal position in the triple mutant context than in the wild
type.

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Fig. 3.
Superposition of binding pockets of the wild
type (yellow) and mutant (gray)
structures. The estradiol A ring superposes perfectly in
both structures, whereas the D-ring is slightly
shifted.
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Effect of C381S Mutation on H12-Core Interface--
The cysteine
mutation at position 381 induces a destabilization of the agonist
position of H12, which is most likely due to a solvating effect. This
residue is located in helix H4, and its side chain is directed toward
the solvent and is located in the agonist binding groove of H12. This
residue is accessible in the mutant structure where H12 is in the
antagonist position. In the wild type structure the cysteine residue is
precluded from the solvent by helix H12. In the present structure this
residue, which is now serine, is still solvent-accessible and is
involved in a water-mediated hydrogen bond network lining the helix H12
agonist binding groove. In the mutant receptor, a positioning of H12 in the agonist groove is possible but would require the desolvation of the
serine residue, a process more energetically costly for a serine than
for a cysteine.
Effect of C530S Mutation on Helix H11--
The C530S mutation
disrupts the hydrophobic contact between Cys530 and
Tyr526 (Fig. 5b) and contributes to the
shortening by one turn of helix H11 at its C terminus end, compared
with the wild type structure. A serine residue exhibits different
solvating properties and favors a coil structure with a surface-exposed
side chain, as observed in the Cys
Ser triple mutant. The
shortening of H11 and the subsequent lengthening of loop 11-12 allow
H12 to reach the coactivator binding groove, as observed in the
tamoxifen-ER complex.
Effect of C417S Mutation on Helix H3--
In the wild type
structure, Cys417 is located in the rather flexible loop
6-7. Its side chain is involved in numerous hydrophobic contacts with
neighboring residues (it forms van der Waals contacts with
Phe337 inside an hydrophobic core composed of the
N-terminal parts of H3 (Phe337, Leu345) and of
the
-sheet (Leu410, Leu408; Fig.
5a). The substitution by a serine residue, by disrupting these hydrophobic contacts, is likely to be the triggering factor that
induces the shortening of helix H3 by one turn at its N terminus. The
conformational reorganization includes the last 10 residues of loop
1-3. Interestingly, the tamoxifen complex is nearly identical to the
triple mutant in this region, whereas the raloxifen-bound structure
exhibits a wild type conformation without shortening. Note that the
overall Cys
Ser triple mutant structure is closer to that of the
tamoxifen-bound LBD than to the raloxifen one. Nevertheless
some differences remain between the mutant and tamoxifen structures,
especially in the loop 6-7 region, which is shifted by more than 3.0 Å (Glu419 and Gly420) toward the core of the
protein. This large movement in the antagonist structure is most likely
induced by tamoxifen, whose aromatic ring is almost perpendicular to
the estradiol D-ring and superimposes on the position
17. This movement of loop 6-7,
filling the cavity, encroaches on the N terminus of helix H3, which
thus adopts a conformation similar to that of the mutant protein.

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Fig. 4.
Additive effect of Cys Ser mutations on the transcriptional activity of the full-length
ER. The graph represents dose-response curves of
transactivation expressed in relative CAT activity as a function of
estradiol concentration. The curve for wild type is in
black, all three single mutations are in green,
the double mutation is in blue, and the triple mutant is in
red.
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The Antagonist Conformation Disrupts a Hydrogen-bonding Network
Observed in Agonist Complexes--
The shortening of helices H3 and
H11 affects the surrounding structure, in particular a hydrogen bond
network present in the wild type involving the conserved
Glu419 on the loop H6-H7, Lys531 on the end of
H11, and Glu339 at the N-terminal part of helix H3 (Fig.
5c). The disruption of these
interactions is a direct consequence of both C417S and C530S mutations.
The triple mutant E339A,E419A,K531A, which is unable to form the
hydrogen-bonding network, further underlines the importance of this
network for H12 positioning. We have analyzed the transcriptional
capacity of this Ala mutant and compared it to the Cys
Ser
mutations using GAL-ER constructs (Fig. 2b). The Ala triple
mutant displayed 62% activity compared with the wild type receptor,
which is comparable with the value of 64% observed with the
C417S,C530S double mutant. The similarity of effects suggests the
occurrence of similar structural features for H12 positioning.

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Fig. 5.
a, effect of C417S mutation on
H3. Superposition of wild type (yellow) and Cys Ser
triple mutant (gray) emphasizing the shortening of H3 by one
turn and the significant conformational change of the loop 1-3 are
shown. b, effect of C530S mutation on H11. Superposition of
wild type (yellow) and Cys Ser triple mutant
(gray), showing the shortening of H11 on the mutant protein
is shown. c, superposition of wild type (yellow)
and triple mutant (gray) ER LBD structures near the mutated
residues. The ligand is anchored by His524 that interacts
with the carboxyl group of Glu419, a residue from L5-6.
This glutamate contacts both the N-terminal end of H3
(Glu339) and the C-terminal end of H11
(Lys531). The hydrogen bond network connecting the
estradiol O17, His524 and Glu419,
Glu339, Lys531 in the wild type structure is
shown. The effect of the C417S and C530S mutations are to shorten by
one turn the N-terminal end of H3 and the C-terminal end of H11,
respectively. This leads to the disruption of the hydrogen bond
network. To confirm the relevance of this network, Glu339,
Glu419, and Lys531 were mutated in alanines and
compared with Cys Ser mutant receptor in transactivation
assays.
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DISCUSSION |
One generally accepted mechanism of antagonism is that steric
hindrance inhibits the natural agonist conformation and favors an
alternative position for H12 that then occupies the binding site of
coactivators in the H3/H4 cleft. The present study allows us to
dissociate the steric effect from the others. Indeed the crystal
structure of the Cys
Ser triple mutant LBD bound to estradiol
adopts a typical antagonist conformation, whereas estradiol binding and
transactivation are not impaired. Each single Cys
Ser mutation
lowers the transcriptional activity by ~20%, showing that the
structurally unrelated regions that are perturbed are equally important
for ER
to fully respond to estradiol stimulation. The combination of
these mutations results in an additive effect in the reduction of
transactivation, leading to a triple mutant that is only half as potent
as the wild type receptor. This cumulative effect of the Cys
Ser
mutations further confirms the lack of cooperativity of the observed
conformational changes. The higher amount of estradiol needed to
stimulate transcriptional activity, compared with the lower
concentration of raloxifen required to repress it, suggests
that Cys
Ser mutations favor the switching of helix H12 toward a
nonproductive conformation. These results favor a dynamic model where
H12 occupies two more or less favorable states, with the mutations or
the ligand affecting the equilibrium. This H12 flexibility is also
illustrated by the ER
bound to genistein complex (15). Genistein
distinguishes itself from SERMs by its smaller size, allowing its total
burying in the ligand binding cavity. In the crystal structure, helix
H12 adopts a position shifted by 25° from the antagonist conformation
observed in the raloxifen complex. Taken together with the
highly disordered helix H12 in the ER
-raloxifen crystal
structure and similar observations in other systems (RXR-RAR), it is
clear that antagonist positions are not unique. The RXR
F318A
LBD-oleic acid complex (34), with a typical antagonist conformation for
H12, bears even more resemblance to our present situation in that the
ligand also does not prevent the agonist position. In this case too the
antagonist conformation is in apparent contradiction with the
transcriptional activity of the receptor and its ability to recruit
coactivators (34). Furthermore an agonist position with a weak partial
agonist is observed in the PPAR
structure (35). All these cases can be explained by a "flip-flop" mechanism for H12 positioning, the equilibrium between the H12 agonist and antagonist positions in the
coactivator binding groove would then depend on the cellular context
(nature and concentration of cofactors) (5).
The comparison of the wild type and the Cys
Ser triple mutant
structures reveals the molecular interactions that are essential for
stabilizing the agonist conformer. If these are weakened or disrupted
by altering the protein hydration and/or conformation, the equilibrium
is shifted toward an alternative nonproductive conformation, explaining
the partial decrease in transcriptional ability. When estradiol binds
to ER, it interacts through the 17
-hydroxyl group with
His524, which in turn forms a hydrogen bond with the
peptidic carbonyl group of Glu419 in the loop 6-7. This
glutamic acid contacts Glu339 from helix H3 and
Lys531 from helix H11, forming a hydrogen bond network that
favors the helix H12 agonist position (Fig. 5c). The loop
1-3 accompanies the movement of helix H3, as shown in other
nuclear receptors. The precise positioning of H3 is an
important feature for the constitution of the ligand binding cavity.
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CONCLUDING REMARKS |
The destabilization of the H12-protein core interaction is at the
heart of the mechanism of partial and pure antagonism. The dominant
effect depends on the potency of the ligand to disrupt the active
conformation or in other words to prevent the correct binding of
coactivators. The present study sheds light on the molecular mechanism
and the structural basis of partial agonism on the AF2 transactivation
function. The position of H12 can be modulated by the cellular context
of cofactors, their ability to displace the equilibrium and to
stabilize one conformer.
For the glucocorticoid and estrogen receptor, cysteine modification
plays a role in gene regulation by the intracellular redox potential
modification (36-38). The importance of cysteine residues located in
the activation domain has recently been stressed for the nuclear factor
I/CCAAT transcription factor (NFI/CTF) (39). Intracellular thioredoxin
or metal ion concentration, which have high affinity for sulfhydril
groups, would act as the regulator of the transcription. Their effect
on transactivation could be explained by the modification of cysteines,
linking hormonal and redox signaling pathways.
ER, like other steroid nuclear receptors, is unstable in the absence of
ligand or protein cofactors like HSP90 (33). The fold stabilization of
these proteins is part of the control of gene expression and is
ligand-dependent (induced fit mechanism) and controlled by
the cellular context (redox potential, nature of a ligand, presence of
interacting molecules like coactivators or corepressors).