(Received for publication, September 27, 1996, and in revised form, November 26, 1996)
From the Department of Physiology and Biophysics, University of California, Irvine, California 92697
The estrogen receptor dimerizes and exhibits
cooperative ligand binding as part of its normal functioning.
Interaction of the estrogen receptor with its ligands is mediated by a
C-terminal hormone-binding domain (HBD), and residues within the HBD
are thought to contribute to dimerization. To examine dimer
interactions in the isolated HBD, a human estrogen receptor HBD
fragment was expressed in high yield as a cleavable fusion protein in
Escherichia coli. The isolated HBD peptide exhibited
affinity for estradiol, ligand discrimination, and cooperative
estradiol binding (Hill coefficient ~1.6) similar to the full-length
protein. Circular dichroism spectroscopy suggests that the HBD contains
significant amounts of -helix (~60%) and some
-strand (~7%)
and that ligand binding induces little change in secondary structure.
HBD dimer dissociation, measured using size exclusion chromatography,
exhibited a half-life of ~1.2 h, which ligand binding increased
~3-fold (estradiol) to ~4-fold (4-hydroxytamoxifen). These results
suggest that the isolated estrogen receptor HBD dimerizes and undergoes conformational changes associated with cooperative ligand binding in a
manner comparable to the full-length protein, and that one effect of
ligand binding is to alter the receptor dimer dissociation kinetics.
The estrogen receptor is a member of a superfamily of nuclear
proteins that includes the receptors for the steroid hormones, for
vitamins A and D, and for thyroid hormone (1, 2). The binding of
ligands to these receptors is the initial step in a complex series of
events culminating in an interaction of the ligand-bound receptor with
the transcription machinery and modulation of gene expression. These
receptor proteins exhibit four distinct properties required to exert
their actions: hormone binding, multimeric complex formation, sequence
specific DNA binding, and transcriptional modulation. The currently
proposed schematic structure of these receptor proteins (shown in Fig.
1 for the estrogen receptor), based on sequence similarities and
deletion analyses (summarized in Ref. 2), suggests that these proteins
fold into at least three separate structural and functional domains:
(i) an N-terminal domain having a highly variable length and amino acid
sequence and believed to mediate much of the transcriptional
enhancement activity of the protein, (ii) a highly conserved central
domain of ~80 amino acids involved in DNA binding, and (iii) a less
well conserved C-terminal domain of ~250 amino acids that is involved in ligand binding.
The C-terminal hormone-binding domain (HBD)1 of the receptor is of particular interest because, in addition to its ligand binding activity, it appears to contain many of the regulatory functions of the protein. Chimeric constructs containing fusions of fragments of the estrogen receptor with unrelated proteins such as the myc oncogene product, for example, display hormonal regulation of the activity of the fused gene products (3). This suggests that, even when removed from its normal environment, the HBD is not only capable of specific ligand binding, but may also retain the capacity to undergo the conformational changes that normally regulate the function of the receptor.
The nuclear receptor superfamily proteins are thought to form dimers. This property has at least two functional roles: most of the proteins in the family are thought to bind DNA as dimers, and dimer formation allows cooperative ligand binding, thereby narrowing the ligand concentration range required for full biological effect. The nature of the dimer interface of the full-length receptor protein is not established. The HBD is thought to play a role in dimerization, since mutations of residues within the estrogen receptor HBD have been shown to inhibit dimer formation (4). The isolated estrogen receptor DNA-binding domain has been shown to dimerize in the presence of DNA, suggesting that some of the dimerization interface resides within this portion of the protein; the isolated DNA-binding domain, however, is monomeric in solution (5, 6). It is not known how much of the interprotein interaction involved in dimer formation is actually contained within the HBD and whether the isolated HBD is fully capable of cooperative ligand binding.
Characterization of the estrogen receptor HBD has been limited by the
difficulty of obtaining sufficient amounts of the peptide. In order to
obtain preparations of the estrogen receptor HBD for more detailed
study, several groups have attempted to express peptides containing the
HBD in heterologous systems. Expression of the estrogen receptor HBD in
yeast and bacteria has generally resulted in low yields of protein:
~1 mg/liter (7, 8). Recently, Seielstad et al. (9)
reported expression of an isolated HBD fragment in high yields in
Escherichia coli; however, the protein produced using this
system was insoluble, necessitating the use of urea during purification
and characterization.
We describe herein a system which yields high level expression of soluble human estrogen receptor HBD in E. coli. The HBD peptide is produced as a fusion protein with the E. coli maltose-binding protein at levels of ~10% of the total cell protein, and the fusion protein can be chemically cleaved to afford micromole quantities of the HBD peptide. We have characterized the cooperativity of estradiol binding and have examined the kinetics of dimer dissociation in solution.
Restriction endonucleases and other enzymes used for DNA manipulation were obtained from Boehringer Mannheim, New England Biolabs, Inc., Stratagene Cloning Systems (La Jolla, CA), or U. S. Biochemical Corp. Synthetic oligonucleotides were obtained from Operon Technologies (Alameda, CA). Bacterial growth media components were purchased from Difco (Detroit, MI); other reagents were obtained from Sigma. Tritiated estradiol was obtained from Amersham and DuPont NEN. The estrogen antagonist trans-4-hydroxytamoxifen was a gift from Dr. Dominique Salin-Drouin (Laboratories Besins-Iscovesco) and ICI 182,780 was a gift from Dr. Alan Wakeling (ICI Pharmaceuticals).
Vector ConstructionUnless otherwise noted, all DNA
manipulations were carried out by standard techniques (10). A DNA
fragment coding for the human estrogen receptor hormone-binding domain
(amino acids 301-551) was generated by PCR from the HE0 estrogen
receptor cDNA plasmid (11) using the following primers: 5
primer TCTAAGAAGAACAGCCTGGCCTTG, and 3
primer
atcCgAaTtcaCGCATGTAGGCGGTGGGCGTCCAG; lowercase bases in the 3
PCR primer are mismatches that convert the codon for Pro-552 to a TGA
termination codon and create an EcoRI site for subcloning.
The PCR fragment was digested with EcoRI and subcloned into
the pMAL-c2 vector (New England Biolabs) which had been digested with
XmnI and EcoRI. Following isolation of the
insert-containing plasmid, the entire HBD coding region was sequenced
to confirm the absence of errors introduced by PCR amplification. The
presence of the cDNA mutation G400V (12) was verified by DNA
sequencing; this mutation was reverted to wild-type using a PCR
mutagenesis procedure (13), creating the plasmid pMAL-HBD1 (Fig.
1).
Protein products of pMAL-c2 derived plasmids consist of the
maltose-binding protein fused to the desired protein with a linker peptide consisting of (Asn)10-Leu-Gly-Ile-Glu-Gly-Arg; the
terminal four residues of the peptide comprise a Factor Xa
cleavage signal. Factor Xa hydrolysis of the expressed
fusion protein, however, resulted in heterogeneous, largely inactive
peptides; we therefore modified the linker region to generate the
sequence Asn-Gly, which can be cleaved by hydroxylamine (14). Bases
encoding residues Leu-Gly-Ile-Glu of the Factor Xa
recognition sequence were mutated to Asn codons by site-directed
mutagenesis using the unique site-elimination procedure (15) with the
Transformer kit from Clontech. The coding region of the mutagenesis
product was sequenced; the modified DNA was found to encode a linker
peptide of (Asn)14-Gly-Arg. This plasmid was designated
pMAL-HBD2. Since N-terminal sequence analysis of the cleaved protein
revealed that approximately 10-40% (the proportion varied in
different preparations) of the protein was also cleaved between Asn-304
and Ser-305 of the HBD sequence (note doublet in Fig. 2, Lane
8), unique site-elimination was performed on pMAL-HBD2 to mutate
Ser-305 Glu, creating pMAL-HBD3. Fig. 1 shows the protein sequences
surrounding the junction between the MBP and HBD for the three
expression plasmids. The product of hydroxylamine cleavage of the
fusion protein from the HBD2 and HBD3 constructs retains Gly-Arg from
the linker, the latter of which corresponds to the naturally occurring
Arg-300.
Protein Expression and Purification
Competent TOPP2 cells
(Stratagene) were transformed with the expression plasmids pMAL-HBD1,
pMAL-HBD2, or pMAL-HBD3. Cells containing the plasmid were grown in TB
media in the presence of 100 µg/ml ampicillin to an OD600
of ~1.7; protein expression was induced by the addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 0.25 mM and cultures were grown overnight at ambient temperature (usually ~27 °C). Cells were resuspended in
lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 2 mM dithiothreitol, 1 mM AEBSF (Cal Biochem), pH
8.0, and 1 mg of lysozyme/g of cells). After ~1 h at ambient
temperature, MgCl2 was added to a final concentration of
120 mM and the lysate treated with DNase and RNase. The
supernatant from a 40,000 × g centrifugation of the lysate was diluted 3-fold in TED buffer (20 mM Tris-HCl, 1 mM EDTA, and 1 mM dithiothreitol, pH 7.3) and
applied to a DEAE-cellulose column (Whatman). The flow-through from the
DEAE-cellulose column was applied to an amylose resin column (New
England Biolabs). After washing with 2-4 column volumes of TED
containing 0.2 M NaCl, the fusion protein was eluted with
10 mM maltose in the same buffer.
The eluted protein was diluted 5-fold and applied to a DEAE-Sepharose
column (Pharmacia). This column was washed with 5 column volumes of TED
containing 0.05 M NaCl, and the protein was eluted with a
linear NaCl gradient (0.05-0.2 M NaCl); the fusion protein eluted at 0.13-0.16 M NaCl. The fusion protein was then
concentrated to ~20 mg/ml by precipitation with 60% ammonium sulfate
and was digested for 60-72 h at ambient temperature with hydroxylamine (final concentration: 2 M hydroxylamine-HCl, 0.2 M Tris-HCl, pH 9.0). The cleaved HBD peptide was separated
from the maltose-binding protein by Sephadex G-100 gel filtration
chromatography. The final preparation of the purified HBD peptide was
stable and could be stored at 4 °C or 70 °C for several
months.
It should be noted that early preparations of the fusion protein and of the HBD had apparent estradiol binding stoichiometries significantly lower than 1:1, although the other properties of the protein were similar to those reported here. Addition of the DEAE-Sepharose chromatography step to the purification procedure for the fusion protein raised the stoichiometry of estradiol binding to close to 1:1, although this step had little effect on the apparent purity as assessed by SDS-PAGE. In contrast, FPLC Superdex-200 or gravity Sephadex G-100 gel filtration chromatography had no effect on the activity of the fusion protein or HBD samples.
SpectroscopyAll spectroscopy was performed at ambient
temperature. Absorbance spectra were obtained using a Cary 1 spectrophotometer calibrated with K3Fe(CN)6
assuming 420 = 1,020 (M cm)
1.
The concentration of purified MBP-HBD fusion protein and isolated HBD
peptide were determined spectrophotometrically assuming
280 = 89,365 (M cm)
1 for the
fusion protein and 23,745 (M cm)
1 for the HBD
peptide; these values are based on a composition of 11 tryptophan and
20 tyrosine residues (fusion protein) or 3 tryptophan and 5 tyrosine
residues (HBD peptide) predicted from the cDNA sequence and on
average extinction coefficients for tryptophan (5615 (M
cm)
1) and tyrosine (1380 (M
cm)
1) (16, 17). Concentrations of HBD determined
spectrophotometrically agreed closely with those determined by the
methods of Lowry et al. (18) and Bradford (19).
Circular dichroism spectra were obtained using a Jasco J-720 spectropolarimeter with a 0.05-cm path length cell and a band pass of 2 nm. Twelve scans were collected and averaged. Theoretical curve fitting to estimate secondary structure content was performed using the k2d program (20).
Analytical Gel FiltrationThe apparent molecular weight of the fusion protein and HBD were determined using a Pharmacia FPLC system and a Superdex 200 HR 10/30 gel filtration column (running buffer 20 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, pH 7.3). The column was calibrated using blue dextran to determine the void volume and with the following standard proteins: thyroglobulin (669 kDa), ferretin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (69 kDa), ascorbate peroxidase (57.5 kDa), P450eryF (45.8 kDa), ovalbumin (43 kDa), MBP (40.4 kDa), rhodanese (33.3 kDa), chymotrypsinogen (25 kDa), ribonuclease A (13.7 kDa), and cytochrome c (12.4 kDa).
For kinetic experiments, equimolar amounts of the fusion protein and
HBD peptide were mixed and incubated at ambient temperature (~25 °C). At various times aliquots were taken and subjected to FPLC gel filtration. For the experiments in the presence of ligand, the
column was pre-equilibrated in the same running buffer with 50 nM of the relevant ligand, and 2 µM solutions
of each protein pre-equilibrated overnight with 5 µM of
the ligand. The integrated peak areas were corrected for extinction
coefficient of the relevant protein species to determine the
concentration of each species (i.e. fusion homodimer, HBD
homodimer, or heterodimer) present at the time of injection (the
relative amount of each species was assumed not to change during the
chromatography). For experiments in the presence of ligand, the
extinction coefficient of the protein was corrected for contributions
of the bound ligand (assumed to be ~2,000 (M
cm)1 for estradiol and ~15,000 (M
cm)
1 for 4-hydroxytamoxifen).
The rate constant for dissociation, k, was determined by least-squares nonlinear regression of the first-order rate equation,
![]() |
(Eq. 1) |
Amino-terminal sequence data was obtained for purified cleaved protein by automated Edman degradation performed by Dr. Agnes Henshen-Edman (University of California, Irvine).
Radioreceptor AssayDilutions of the HBD peptide were
incubated overnight with various concentrations of
[6,7-3H]estradiol at 4 °C in TED buffer including 0.2 M NaCl and 1 mg/ml porcine gelatin; bound and unbound
steroids were separated using dextran-coated charcoal (0.625%
charcoal, 0.125% dextran) in the same buffer without gelatin. In all
experiments using purified and partially purified protein, the binding
of radioactive estradiol in the presence of a 100-fold excess of
unlabeled estradiol was equivalent to the nonspecific binding observed
in the absence of any added HBD protein. The presence of a carrier
protein in both the ligand and protein buffers was found to be
necessary to obtain reproducible results; porcine gelatin (1 mg/ml),
bovine -globulin (4 mg/ml), or bovine serum albumin (4 mg/ml) gave
similar results.
The data for bound and free steroid were directly fitted to the Hill equation (21),
![]() |
(Eq. 2) |
For competitive ligand binding experiments, the HBD peptide was incubated with various concentrations of ligand in the presence of a constant amount of tritiated estradiol. The amount of bound [3H]estradiol is presented as a percent of that bound in the absence of competitor ligand.
The pMAL-c2 expression system produces the protein of interest as a fusion with the E. coli maltose-binding protein. The MBP is well expressed, stable, and can be purified by amylose affinity chromatography. The linker peptide of the fusion protein is designed to be cleavable by the endoproteinase Factor Xa to release the fused protein without additional N-terminal residues. The construction of vectors for expressing the MBP-HBD peptide is shown schematically in Fig. 1, and described under "Materials and Methods."
Induction of protein expression from each of the pMAL-HBD plasmids with
isopropyl-1-thio--D-galactopyranoside produced
significant quantities of fusion protein, estimated to comprise
approximately 10% of the cell protein (Fig. 2,
Lane 2). Essentially all of this fusion protein appeared to
be soluble in the cells. Attempts to purify the MBP-HBD fusion from
crude cell homogenates by amylose affinity chromatography, however,
were unsuccessful; binding of the fusion protein from the crude extract
to the affinity column was incomplete, and the eluted protein was not
highly purified. For this reason, the cell lysate was first subjected
to anion exchange chromatography. The partially purified fusion protein in the unbound fraction from the anion exchange column bound the amylose column nearly quantitatively, and upon elution from the column
exhibited only minor contaminants (Fig. 2, Lane 3).
Enzymatic cleavage of the partially purified MBP-HBD1 fusion protein with either Factor Xa or trypsin resulted in degradation to heterogeneous products that exhibited markedly reduced ability to bind estradiol (data not shown). Chemical cleavage methods were then tested as alternatives to proteolytic digestion. Hydroxylamine preferentially hydrolyzes the peptide bond of Asn-Gly sequences, although solvent-exposed Asn-Xaa sequences are hydrolyzed at lower rates (14). The HBD peptide does not contain any Asn-Gly sequences and would therefore be expected to be relatively resistant to hydroxylamine. Modifications to the original construct resulted in the expression plasmid pMAL-HBD3 (Fig. 1); the fusion protein from this plasmid appeared to cleave quantitatively cleavage at the correct site, yielding a final peptide with Gly followed by amino acids 300-551 (S305E) of the HBD (see Fig. 2, Lane 4, for the results of the cleavage reaction).
The HBD peptide was separated from the MBP by gel filtration chromatography. Analysis of a final preparation of the HBD peptide by SDS-PAGE is shown in Fig. 2, Lanes 5-7. The HBD peptide has an apparent mass similar to the predicted ~29 kDa. Based on the staining intensities observed the overall purity is estimated to be >90%; the minor impurity band visible at the highest concentration of HBD peptide is estimated to comprise ~1-2% of the total protein (based on densitometry of the Coomassie-stained gel), and is a degradation product of the HBD peptide. The final yield was typically ~10 mg of purified HBD peptide per liter of bacterial culture for several preparations; this corresponds to approximately a 40% yield from the total amount of fusion protein estimated to be present in the cells.
Ligand BindingBoth the purified HBD peptide and the MBP-HBD fusion protein were assayed for their ability to bind estradiol. Fig. 3 shows the results of typical Scatchard analyses of [3H]estradiol binding using low concentrations (0.15 nM) for each protein. The Kd values obtained (0.1 nM) are similar to those reported for the full-length estrogen receptor protein obtained from human cells (cf. Ref. 8) indicating that the estrogen binding properties of the HBD are not affected either by isolation from other parts of the receptor or by fusion to the maltose-binding protein. The Bmax values determined in this experiment corresponded to a binding stoichiometry of ~0.98 mol of estradiol bound per mol of HBD peptide. No significant changes in stoichiometry have been observed following the hydroxylamine cleavage step, suggesting that little denaturation occurs under the conditions of the cleavage procedure.
Ligand Discrimination by the HBD PeptidesThe ability of the
HBD peptide to discriminate between different ligands was assessed by
competitive binding assays using [3H]estradiol, and the
competition binding curves are presented in Fig. 4. The
ligand discrimination profile exhibited by the isolated HBD peptide is
generally similar to that reported for the full-length native receptor
(8). The weak agonist estrone exhibited about 10-fold lower affinity
than estradiol, whereas testosterone (and progesterone, data not shown)
did not appear to compete significantly even at concentrations
35,000-fold greater than those used for estradiol. The steroidal
antagonist ICI 182,780 bound with an affinity intermediate between that
of estradiol and estrone. Two non-steroidal antagonists,
trans-tamoxifen and trans-4-hydroxytamoxifen,
were tested and also found to be effective competitors. The
trans-4-hydroxytamoxifen had a slightly lower affinity and a
steeper slope on the semi-log plot than that of estradiol, similar to
observations previously reported by Sasson and Notides (23) for the
full-length calf uterine estrogen receptor. The non-parallel
competition curve for 4-hydroxytamoxifen was interpreted by Sasson and
Notides (23) to indicate that estradiol and 4-hydroxytamoxifen bind the
receptor differently, and that 4-hydroxytamoxifen binding to one site
in the dimer induced the dissociation of estradiol from the other site.
Our observation of the non-parallel competition of estradiol by
4-hydroxytamoxifen suggests that the structural features required for
this differential binding of the two ligands are retained by the
isolated HBD.
Circular Dichroism Spectra of the Purified HBD Peptide
Far
ultraviolet circular dichroism was used to investigate the peptide
backbone secondary structure (Fig. 5). The spectrum exhibits a maximum near 195 nm, and minima at 208 and 222-223 nm.
Curve-fitting to the data using the k2d computer program (20) suggests
a composition of ~60% -helix and ~7%
-strand. Both secondary structure prediction methods (24, 25) and the known crystal
structure of the related retinoid-X-receptor-
(RXR-
), retinoic
acid receptor-
(RAR-
), and thyroid hormone receptor-
HBDs
(26-28) predict a largely helical fold, and the latter proteins have
similar helical character (60-65%) and similar amounts of
-strand
(5-10%). CD spectra were also recorded for the HBD in the presence of
equimolar amounts of the ligands estradiol and trans-4-hydroxytamoxifen; these spectra were essentially
identical to the spectrum in the absence of ligand (Fig. 5), suggesting that any conformational changes induced by ligand binding do not involve significant changes in overall secondary structure.
Size Exclusion Chromatography
The hormone-binding domain is
thought to contain a region at least partially responsible for
dimerization of the full-length protein (2, 4, 29). We used
gel-filtration chromatography to determine whether the fusion protein
and HBD peptide formed dimers (or larger multimers) in solution. When
the cleaved HBD peptide was separated from the MBP by Sephadex G-100
gel filtration chromatography during the purification procedure, the
HBD peptide was found to elute near the void volume of the column, well
ahead of the ~40-kDa MBP. This suggested that the HBD peptide
(monomer ~29 kDa) exists as a multimeric complex under the conditions
used for the preparative G-100 gel filtration (~100 µM
initial peptide concentration). This was examined further using
analytical FPLC Superdex-200 gel filtration chromatography. When run
independently on the Superdex column, both the fusion protein and HBD
peptide migrated as single peaks. The apparent
Mr of the fusion protein (156,000) was similar
to that predicted for a dimer (142,000). Because the isolated MBP is
known to be monomeric and migrates close to its predicted size of
40,000, this suggests that fusion protein dimerization is mediated by
the HBD peptide fragment. The HBD peptide alone migrated with an
apparent Mr of 47,000, intermediate between that
expected for a dimer (58,000) or monomer (29,000). To determine whether
the HBD peptide exists as a dimer, equimolar amounts of the fusion
protein and HBD peptide were mixed, allowed to equilibrate for 24 h, and then subjected to gel filtration. Under these conditions, a
third peak appeared at 99,000 (Fig. 6), a position
corresponding to a heterodimer of the fusion protein (72,000) and the
HBD peptide (29,000). Integration of the peak areas (corrected for the
extinction coefficients of fusion homodimer, heterodimer, and HBD
peptide homodimer) yielded a ratio of 1:2:1, confirming the identity of
the intermediate peak as a heterodimer formed between fusion protein
and HBD peptide monomers. The fact that a single additional peak was
formed also suggests that both fusion protein and HBD peptide
predominantly form dimers, but not larger multimers, in solution. No
significant changes were observed in the migration of the HBD peptide
or fusion protein on the Superdex column using concentrations ranging
from 0.5 to 10 µM. This suggested that for both the
fusion protein and HBD peptide, the majority of the protein was present
as dimer under these conditions.
In order to form the heterodimer, these homodimers must dissociate, and
this dissociation probably constitutes the rate-limiting step for
heterodimer formation. The fusion protein and HBD peptide were
therefore subjected to gel filtration at various times after mixing. A
plot of homodimer concentration (determined from peak area)
versus time after mixing fits a first-order exponential (Fig. 7); the rate constant for dissociation was
determined to be 0.60 ± 0.14 h1, corresponding to a
half-life of 1.2 h.
We also studied the effects of ligand binding on the dimer dissociation. For these experiments the protein was pre-equilibrated with saturating amounts of estradiol or 4-hydroxytamoxifen. These results, also shown in Fig. 7, are summarized in Table I. The data suggest that presence of estradiol significantly decreased the rate of dissociation of the receptor dimer. The antagonist ligand trans-4-hydroxytamoxifen was also found to significantly decrease the rate of dissociation, to an even greater degree than estradiol.
|
The full-length native estrogen receptor protein has been shown to exhibit positive cooperativity (30, 31). We measured [3H]estradiol binding using several different concentrations of the HBD peptide to determine whether this aspect of the functional nature of the dimer was retained; the data were analyzed by least-squares nonlinear regression as described under "Materials and Methods." Fig. 8 shows the results of a typical experiment at several concentrations of the HBD peptide. At the lowest concentration shown (2 nM HBD peptide) the best fit to the data has a Hill coefficient of 1.0, yielding a straight line on the Scatchard plot similar to that presented in Fig. 3 in which 0.15 nM HBD peptide was used. In contrast, at the higher concentrations (4-15 nM HBD peptide) the data exhibit convex curvature indicative of positive cooperativity. The inset in Fig. 8 shows a Hill plot of the data from the highest concentration of the HBD peptide; the line determined from this set of data has a slope (Hill coefficient) n = 1.54, indicating positive cooperativity. In a series of these experiments, non-cooperative estradiol binding was observed when the peptide concentration was less than ~2 nM, and maximal cooperativity was observed at concentrations greater than ~3 nM. The observed F0.5 values increased from ~0.1 to ~0.5 nM over the same HBD peptide concentration range. The maximal Hill coefficient observed for the HBD peptide (n = 1.5-1.6) is similar to the value of ~1.6 reported for the full-length receptor (31). Moreover, the concentration range over which ligand binding to the isolated HBD peptide changes from non-cooperative to cooperative behavior (~2-3 nM) is also similar to the range of 1-7 nM over which the full-length human receptor begins to exhibit cooperativity (31). These results suggest that most or all of the structural features required for cooperative ligand binding interactions are retained in the HBD peptide fragment.
We have expressed, purified in high yield, and carried out initial
characterization of the hormone-binding domain of the human estrogen
receptor. The yield of soluble HBD peptide obtained (~10 mg/liter of
culture) is significantly greater than previously reported, and
represents amounts of protein that can be used for a variety of
biophysical studies. Previous attempts at HBD peptide expression in
E. coli (7, 8) used constructs coding for estrogen receptor
peptides comprising amino acid residues 240-595, and therefore
sequences of more than 350 amino acids. Moreover, these expressed
peptides were fused to either a peptide from -galactosidase (7) or
to Protein A (8), and the HBD peptide products were not reported to be
either purified or cleaved from the fusion proteins. Recently,
expression of an HBD peptide fragment (amino acids 282-595) in high
yield in insoluble form from a T7 RNA polymerase expression plasmid was
reported (9). This protein, however, required 1 M urea for
solubilization and 5 M urea and estradiol for purification;
moreover, the preparation was heterogeneous as a result of proteolysis
at positions 569 or 571.2
The expression system we describe herein produces an active fragment of the estrogen receptor comprising only 253 amino acids (positions 300-551, with an additional Gly at the amino terminus and Ser-305 mutated to Glu) following purification and cleavage.3 As discussed in more detail below, the estrogen receptor HBD peptide exhibits ligand binding and ligand discrimination properties comparable to the full-length protein, suggesting that the changes incorporated do not grossly alter the structure of this domain of the receptor. The reduced size of our construct should simplify interpretation of biophysical data regarding the expressed protein. In addition, this peptide is expressed in soluble form at high levels and does not require the use of urea nor estradiol in the purification procedure. It is not clear whether the high level of expression we observe is due to the composition of the estrogen receptor-derived peptide, to the efficiently expressed maltose-binding protein used as a leader, or to a combination of the two.
Properties of the HBD PeptideThe HBD peptides exhibited both
high affinity estradiol binding (Kd ~0.2
nM), and ligand discrimination similar to that of the
full-length receptor. The ligand binding observed is consistent with a
single ligand interacting with a single HBD peptide monomer. The CD
spectrum suggests that the HBD peptide is largely helical, as expected
from both secondary structure prediction and from the crystal
structures of the related (~25% sequence identity) RAR- (26) and
RXR-
(27) and thyroid hormone receptor-
HBD (28) peptides. The
observation that the CD spectrum is essentially unchanged in the
presence of both agonist ligand estradiol and the non-steroidal
antagonist ligand trans-4-hydroxytamoxifen indicates that
the overall amount of secondary structure is unaffected by the
conformational changes that occur upon interaction with either type of
ligand. Comparison of the monomeric agonist-complexed RAR-
and
dimeric ligand-free RXR-
structures shows that the major
conformational changes are confined to movements of one loop and of the
-helix at the C terminus of the HBD peptide (27, 32). Our CD data
provide experimental support for a similar model for the estrogen
receptor, and further suggest that alterations in dimer interactions as
a result of ligand binding also do not involve major changes in
secondary structure.
The estrogen
receptor is thought to bind to DNA as a dimer (2, 4, 29). Mutations
within the C-terminal region of the HBD peptide (positions 507-518 of
the mouse receptor, corresponding to 503-514 of the human estrogen
receptor) have been shown to disrupt dimerization (4, 33). In addition,
the RXR- HBD peptide crystallized as a dimer (26). On the other
hand, the HBD peptides from two other related proteins, RAR-
(27)
and thyroid hormone receptor-
(28), crystallized as monomers.
Although the isolated estrogen receptor DNA-binding domain is monomeric in solution (5), it binds DNA as a dimer (6), raising the question as
to whether interactions involving residues in the HBD peptide alone are
sufficient to allow stable dimer formation. At the high concentrations
used for the preparative G-100 column (~100 µM) and the
intermediate concentrations used for the Superdex S-200 chromatography
(0.5-10 µM), both the fusion protein and HBD peptide
migrated at apparent sizes significantly greater than those predicted
for their respective monomers, and a mixture of fusion protein and HBD
peptide resulted in the appearance of third peak corresponding to a
heterodimer both in the presence and absence of ligand. Our results
thus suggest that the isolated HBD peptide contains the amino acid
sequences sufficient for dimerization, and furthermore, that dimer
formation does not require ligand binding.
Dimer formation was also implied by the finding that the HBD peptide exhibited positively cooperative estradiol binding, with a maximal Hill coefficient of ~1.6. Cooperativity of estradiol binding to the estrogen receptor has been most extensively studied using the protein from calf uterine cytosol (30, 34), and the maximal Hill coefficient cited in these reports is ~1.6 at a concentration of ~5 nM receptor. Using recombinant full-length human estrogen receptor expressed in Sf9 insect cells, Obourn et al. (31) also found a maximal Hill coefficient of ~1.6; their data suggest that at receptor concentrations below ~1 nM, the receptor does not exhibit cooperativity, while maximal cooperativity is observed at 10-20 nM receptor concentration. We observe a transition from non-cooperative to cooperative behavior for the isolated HBD fragment in the range from 2 to 3 nM, suggesting that the HBD peptide undergoes the conformational changes required for cooperativity in a manner comparable to the full-length protein. One interpretation of these results is that dimer formation occurs over this peptide concentration range in the presence of estradiol. Thus, at concentrations below ~2 nM, the HBD peptide exists as a monomer, while at concentrations above 3 nM, the HBD peptide is predominantly present as a dimer which exhibits a cooperative interaction with estradiol.
The ligand-bound RAR and thyroid hormone receptor structures were determined for monomeric proteins, and therefore do not allow conclusions to be drawn as to the structural effect of ligand binding on dimer interactions. The cooperativity data suggest that conformational changes in one subunit of the dimer affect the other subunit. In order to examine the effect of ligand binding on dimer interactions more directly, we took advantage of the significant difference in size between the fusion protein and HBD peptide. We used size-exclusion chromatography to examine the exchange from homodimers to heterodimers of one fusion and one HBD monomer. In the absence of ligand, the dissociation (presumed to be the rate-limiting step in the exchange process) was slow, with a half-life of ~1.2 h. In the presence of estradiol the half-life increased significantly by a factor of ~3-fold. The change in dissociation kinetics suggests that ligand binding results in a conformational change in the HBD that affects the dimer interface. The fact that the antagonist 4-hydroxytamoxifen also decreases the rate of dissociation implies that it induces conformational changes in the dimer interface similar to those of estradiol. The increase in half-life suggests that one role of ligand binding is to increase the kinetic stability of the estrogen receptor dimer-ligand complex. One effect of this may be to increase the time available for the receptor dimer to interact with the other proteins of the transcription initiation complex.
We thank Dr. Dominique Salin-Drouin for supplying trans-4-hydroxytamoxifen, Dr. Alan Wakeling for his gift of ICI 182,780, and Dr. Jill Cupp-Vickery for development of the procedure for the hydroxylamine cleavage.