Full text of DOI:10.1093/nar/28.13.2494

2494–2502 Nucleic Acids Research, 2000, Vol. 28, No. 13
© 2000 Oxford University Press
Quantitative characterization of the interaction between
purified human estrogen receptor α and DNA using
fluorescence anisotropy
Mireille Boyer, Nicolas Poujol, Emmanuel Margeat and Catherine A. Royer*
Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier cedex, France
Received March 23, 2000; Revised and Accepted May 16, 2000
ABSTRACT
or the vitamin D, retinoic acid and retinoid X receptors. These
proteins are made up of an N-terminal transactivation domain, a
central DNA binding and dimerization domain, and a C-terminal
ligand binding, dimerization and transactivation domain in
addition to linker regions and determinants for nuclear trans-
location.
In an effort to better define the molecular mechanisms
of the functional specificity of human estrogen
receptor α, we have carried out equilibrium binding
assays to study the interaction of the receptor with a
palindromic estrogen response element derived from
the vitellogenin ERE. These assays are based on the
observation of the fluorescence anisotropy of a
fluorescein moiety covalently bound to the target
oligonucleotide. The low anisotropy value due to the
fast tumbling of the free oligonucleotide in solution
increases substantially upon binding the receptor to
the labeled ERE. The quality of our data are sufficient
to ascertain that the binding is clearly cooperative in
nature, ruling out a simple monomer interaction and
implicating a dimerization energetically coupled to
DNA binding in the nanomolar range. The salt
concentration dependence of the affinity reveals
formation of high stoichiometry, low specificity
complexes at low salt concentration. Increasing the
KCl concentration above 200 mM leads to specific
binding of ER dimer. We interpret the lack of temper-
ature dependence of the apparent affinity as indicative
of an entropy driven interaction. Finally, binding assays
using fluorescent target EREs bearing mutations of
each of the base pairs in the palindromic ERE half-
site indicate that the energy of interaction between
ER and its target is relatively evenly distributed
throughout the site.
Given the involvement of these proteins in a large number of
human pathologies, they have been the target of drug develop-
ment for some time, and anti-estrogens, for example, are widely
used in the treatment of breast cancer. A thorough, quantitative
understanding of the structure–energetics–function relations in
these systems would greatly contribute to the elaboration of
novel therapeutic approaches to the treatment of human path-
ologies involving the estrogen receptor. A number of quantitative
biophysical studies of the interactions between purified full-
length ER and its various biological partners have been
published. For example, Gorski and co-workers (3–7) examined
ER–DNA interactions using pull-down methods. While these
studies yielded precise values for the apparent affinities, they
were carried out using cell extract, rather than purified
receptor, such that extrinsic factors may have participated in
the complexation. Their results (3,5) suggested that estradiol
binding did not affect ER interaction with its EREs and that the
protein bound as a monomer. A certain number of gel retardation
studies have also appeared (i.e., 8–12) in which the interactions
between ER and DNA, the effects of ligand and target
sequence were explored. This method provides interesting
information concerning the number of stoichiometric
complexes formed, but suffers from its non-equilibrium nature
and the relatively low signal-to-noise ratio inherent in the
titration curves derived from quantification of the bands. The
quality of such data is insufficient for determination of the
presence and degree of cooperativity in binding. Recent studies
of ER–ERE interactions based on surface plasmon resonance
suggest ligand dependence in ER–ERE interactions (13) in
contradiction with the earlier work of Gorski and co-workers
(3,5). However, mass transport effects, in addition to the
overall non-equilibrium nature of these measurements, renders
desirable the use of a true equilibrium method for evaluating
the affinity and cooperativity of ER–ERE interactions and for
testing the effects of salt, ligand binding, temperature and
target sequence on these interactions. A series of in-depth
thermodynamic studies on glucocorticoid receptor DNA
binding domain (GR-DBD) interactions with glucocorticoid
and estrogen response elements (GRE and ERE) targets has
INTRODUCTION
The estrogen receptor (ER) is a member of the nuclear receptor
family of ligand activated transcriptional regulators (1,2). In
response to estrogens, this protein activates transcription of a
number of genes in mammalian cells including vitellogenin,
cathepsin D, transforming growth factor α and the oncogene
product c-fos. The action of estrogens has been linked to the
development of uterine, breast and endometrial cancers. ER
shares a strong homology with other members of the nuclear
receptor family, those both closely and more distantly related,
such as the glucocorticoid receptor and the androgen receptor,
*To whom correspondence should be addressed. Tel: +33 4 67 41 79 02; Fax: +33 4 67 41 79 13; Email: royer@tome.cbs.univ-montp1.fr

Nucleic Acids Research, 2000, Vol. 28, No. 13 2495
provided fundamental information concerning the underlying
biophysics of these interactions (14,15). However, it is impor-
tant to address the eventual role of the rest of the nuclear
receptor proteins by comparing results obtained with the
isolated DNA binding domain to those that can be obtained
using the full-length proteins.
sense to 85°C for 10 min in 10 mM Tris buffer in the presence
of 0.1 mM EDTA, 0.1 mM DTT, pH 7.5 (TD buffer) and
cooling slowly to room temperature in a beaker with the
heating water set on the bench. Annealed oligonucleotides
were stored at –80°C.
Ligands
In the present work, we have begun such a biophysical
characterization of ER interactions by carrying out fluores-
cence-based binding assays on the interaction between full-
length baculoviral expressed human ERα and its target DNA
response element. Our equilibrium assays are based on the
observations of changes in the fluorescence anisotropy of a
fluorescein labeled target upon binding by the protein. Because
rotational diffusion of the free oligonucleotide is quite rapid,
the anisotropy of the fluorescent dye covalently bound to the
oligonucleotide is quite low, i.e., little of the orientation of the
polarized exciting light is retained in the emission. However,
because binding by the protein significantly slows the rotational
diffusion of the oligonucleotide, much more of the polarization
of the excitation is retained in the emission. These measure-
ments can be made with very low concentrations of DNA
target and can provide data of very high precision and repro-
ducibility. Thus, they can be used to characterize quantitatively
the affinity, cooperativity and eventually the kinetics of bio-
molecular interactions. Earlier, we reported measurements on
ER–ERE interactions, but the labeling scheme used in these
prior studies did not yield data of the quality required to deter-
mine cooperativity and small differences in affinity (16). Here
we expand upon this earlier work, testing the effects of salt,
ligand, temperature and target sequence on the full-length
human ERα–ERE interactions.
17-β-Estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam) were
purchased from Sigma Chemicals (St Louis, MO) and ICI
182780 (ICI) was a kind gift from Astra Zeneca (London).
Ligands were stored at –20°C in ethanol at 1 mM or diluted
into the TD buffer.
Anisotropy assays
Binding assays were performed using a Beacon 2000 polarization
instrument regulated at the indicated temperature. Target
oligonucleotide concentration was 1 nM. Each point in the
titration curve was obtained by starting with 1 ml of a solution
of 82 nM ER. Aliquots of 200 µl were successively removed
from the starter solution and replaced by 200 µl of buffer
solution containing 1 nM in DNA. The buffer solution was
10 mM Tris–HCl, 0.1 mM EDTA, 0.1 mM DTT, pH 7.5, and
contained the indicated concentration of KCl. Tubes were
equilibrated at the temperature of the measurement for 5 min
prior to measurement and the anisotropy was measured
successively until stabilized. The reported values are the
average of three to five measurements after stabilization.
Data analysis
Binding data of all types were analyzed using the package
BIOEQS, which differs somewhat from the approaches
habitually used. The binding program used by our group,
BIOEQS, was developed in 1991 (17–19) as a means for
analyzing systems that may include a number of oligomeric
protein species, in various states of ligation. In order to easily
analyze data according to complex models and to test various
models incorporating different sets of species, the program
makes use of a numerical, rather than analytical, solver engine.
Instead of deriving an analytical expression for the binding
isotherm in terms of dissociation constants implicit in the
model of choice, the simultaneous set of non-linear free energy
equations associated with the model is solved numerically in
terms of the concentrations of the individual species postulated
to exist. The free energies that are recovered from the fits using
this program correspond to the free energies of formation of
each postulated species (i.e., dimer/DNA complex) from the
free elements (free dimer and free DNA).
The model which was employed to fit the FC6-25mer
profiles corresponds to the case of a system in which a protein
binds to DNA in both a 1:1 and in a 2:1 monomer/DNA
complex (see schematic below). The first free energy in the
model, ∆G°1, corresponds to the free energy of formation of the
1:1 monomer/DNA complex (MDNA) from free monomer (M)
and free DNA, while the second free energy, ∆G°2, corresponds to
the free energy of formation of the 2:1 monomer/DNA
complex (M2ERE), also from free monomer and free DNA. In
order to calculate the free energy of binding of the second
monomer to the 1:1 dimer/DNA complex, ∆G°21, ∆G°1 must be
subtracted from ∆G°2. The cooperative free energy ∆G°coop, is
calculated as the difference between ∆G°2 and two times ∆G°1.
MATERIALS AND METHODS
Protein
Full-length purified baculovirus expressed ERα was purchased
from Panvera corp (Madison, WI). The concentration of active
receptor in each preparation was determined by the supplier by
tritiated estradiol binding and compared to the concentration of
total protein obtained by Bradford analysis. All preparations
were over 80% pure and active. Thus the concentration of
protein was taken to be the concentration of protein capable of
binding estradiol, and not the total protein concentration. The
protein was stored in aliquots of 50 µl at –80°C.
Oligonucleotides
Labeled oligonucleotides were purchased in HPLC purified
form from Eurogentec (Seraing, Belgium) for the internally
labeled target and from Genosys (Montigny-le-Bretonneux,
France) and Genset (Paris, France) for the F-vitERE and
mutant targets respectively. The fluorescein label was incorpo-
rated by the supplier using phosphoramidite chemistry, and all
free probe was thus eliminated in the synthesizer and subse-
quent HPLC purification. The labeling ratio for the sense
strand was calculated using known extinction coefficients for
the four bases and the oligonucleotide composition and a molar
extinction coefficient of 90 000 M-1 cm-1 for the fluorescein.
All oligonucleotides presented labeling ratios between 40 and
80%. The sense and anti-sense strands were annealed by
heating a 1.1 molar ratio of unlabeled anti-sense with labeled

2496 Nucleic Acids Research, 2000, Vol. 28, No. 13
With each marquardt minimization iteration, the solver uses
the current free energy values, along with the mass balance
constraints (i.e., total protein and DNA concentrations) to
calculate the species concentrations using an iterative
constrained optimization routine with the mass balance
constraints incorporated by Lagrange multipliers. The calculated
data are generated from this numerically derived species
concentration vector according to the user specified observable
mapping scheme. In this way, the user must indicate how each
individual species is related to the experimental observable. The
plateau values for the anisotropies were floating parameters in
the fits, while the anisotropy corresponding to the intermediate
monomer-bound DNA species was fixed at a value approxi-
mately half-way between the two plateaus. The details of this
program have been given elsewhere (17–19). Uncertainties on
the recovered parameters were obtained by repeating a
complete minimization over a range of tested parameter
values, allowing all other parameters to float. The reported
errors represent the uncertainties at the 67% confidence limit
(i.e., 1 standard deviation) taking into account the correlation
between all the parameters in the fits.
Figure 1. (a) Fluorescence anisotropy profile of full-length human ERα binding
to 1 nM 5′-fluorescein labeled vit-ERE in 200 mM KCl in TD buffer at 21°C.
The full line through the data points represents the fit to the data using the
cooperative model including a monomer-bound intermediate as described in
the Materials and Methods. The dotted line represents a fit with a simple binding
model. (b) Residuals of the fit to the cooperative (full line) and simple (dashed
line) binding models. The point plotted at the lowest protein concentration is
in fact obtained in the absence of protein.
RESULTS AND DISCUSSION
In earlier collaborative work with the group of J. Gorski (16),
we used a rhodamine labeled target oligonucleotide to study
ER–ERE interactions. However, due to high probe mobility,
and inefficient labeling, the quality of the anisotropy data
obtained in those previous titrations did not permit us to determine
whether the binding of ER to its target ERE was cooperative in
nature. The present studies are designed to provide data of
sufficient quality for establishing whether or not binding is
cooperative and for comparison of binding affinities under a
variety of conditions and for a variety of target sequences. In
order to achieve the necessary precision in the data, we have
used two different labeling schemes for the target oligonucleo-
tides, either through a 5′-phosphoramidite-six carbon linkage
on the sense strand of the target site, or alternatively, incorporated
internal to the sequence in place of the thymine base at position
+5 from the 5′-end of the sense strand. The fluorescent probe
used was fluorescein to maximize the sensitivity of the assay.
Both types of labeling yielded good quality data and binding
constants within error of each other under similar conditions.
The oligonucleotides were 35 bp in length and derived from
the palindromic ERE upstream of the vitellogenin promoter.
The wild-type target sequence referred to here as F-vitERE and
has the sequence given below for the sense strand.
F-5′-AGCTTCGAGGAGGTCACAGTGACCTGGAGCGGATC-3′
F-vitERE
Figure 1a shows the anisotropy-based binding isotherm
obtained upon titration of the 5′-fluorescein labeled wild-type
ERE 5′-F-vitERE with purified full-length human ERα at
21°C in the presence of 200 mM KCl in the TD buffer. No
changes in fluorescence intensity accompanied the increase in
anisotropy, and lifetime measurements on the free and bound
fluorescein labeled oligonucleotide revealed identical 4.3 ns
decay for the free and bound species. The anisotropy increase
obtained under these buffer conditions could be reversed by
addition of unlabeled vit-ERE, but not by addition of poly(dI–dC)
(data not shown), demonstrating the specific nature of the
interaction. The solid line through the points represents a fit of
the anisotropy data in Figure 1a to the model described in the

Nucleic Acids Research, 2000, Vol. 28, No. 13 2497
Materials and Methods. A total of seven binding profiles for
the ER interaction with the F-vitERE target were analyzed.
The average value over the seven data sets for the total
free energy for the binding of two monomers ∆G°2 to the ERE
of –21.8 0.9 kcal/mol and the average value of the ∆G°coop is
found to be 1.7 0.7 kcal/mol. In terms of affinity, one can
calculate an apparent C1/2 (concentration of 50% bound) from
half of the value of ∆G°2. This corresponds to an apparent Kd of
9 nM. There is a reasonably high degree of uncertainty on the
value of the cooperativity since under highly cooperative
conditions the value of ∆G°1 is not well determined because
the intermediate monomer-bound DNA species is never highly
populated.
It can be seen quite clearly from Figure 1a and b that the
cooperative model provides a much better fit to the data
(compare full to dashed lines). This model, implicating an
intermediate monomer-bound species, has been used to
analyze the interaction of the estrogen receptor DNA binding
domain (ER-DBD) with the ERE (14,15). However, in that
case, such a model was clearly justified since the ER-DBD is
monomeric in solution up to millimolar concentrations. In the
case of the full-length ER used here, the concentration dependence
of its oligomeric state has not been determined. We have found
that the isolated hormone binding domain (HBD) of ER
remains dimeric down to a concentration of 1 µM (unpublished
results). We can suppose that the tendency to form dimers
would be greater for the full-length receptor since both the
HBD and DBD dimerization domains are present. The fact that
the protein binds cooperatively to its target demonstrates that
there is protein–protein interaction linked energetically to the
protein–DNA interaction in this system. Although the final
stoichiometry of the complex cannot be unambiguously
deduced from the data in Figure 1a, it is highly likely that it
corresponds to one dimer per target oligonucleotide. We base
this assumption on what is known of the structure of the ER-
DBD–ERE interaction (20) and the small size of the oligo-
nucleotide. We note that the two possible mechanisms of the
cooperativity (intermediate free dimer or intermediate bound
monomer) are equally likely. Only stopped-flow kinetic
measurements in the future will allow the determination of
which of these two mechanisms is operative.
Figure 2. Salt concentration dependence of ER–ERE interactions. (a) Profiles
for titrations of F-vitERE obtained in presence of 150 (open circles), 200
(squares), 250 (triangles) and 300 mM KCl (closed circles); (b) profiles
obtained for F-vitERE at 200 mM KCl (squares), a mutant sequence mutF (see
Fig. 5) at 200 mM KCl (circles) and in the absence of salt (triangles). The profiles
were obtained in TD buffer at 21°C, and the DNA concentration was 1 nM. In
this case the DNA target was labeled on the 5′-end through a six-carbon linker.
Lines through the points represent fits to the data in terms of the model
described in the Materials and Methods. The points plotted at the lowest protein
concentration were obtained in the absence of protein.
Salt effects on ER–ERE interactions
The effect on ER–ERE interactions of varying the concentration
of KCl in the binding buffer at 21°C is shown in Figure 2a and
b. As before (16) we find that decreasing the salt concentration
yields profiles exhibiting a much larger increase in anisotropy,
indicative of the formation of complexes of significantly
higher stoichiometry. At 150 mM, for example, the total
change is 40 mA units, whereas at 200 mM KCl the total
change is only ~32 mA units. Between 200 and 300 mM KCl
the effect of salt on the specific dimer–DNA interaction is
quite large, consistent with the 12 phosphate contact observed
in the crystal structure of the ER-DBD complexed with DNA
(20,21). A similarly strong salt concentration effect has been
observed for the GR-DBD/GRE interaction (22, J.J.Hill,
C.A.Royer and K.R.Yamamoto, unpublished results). We note
that the loss of cooperativity at higher salt concentration arises
because the ER is likely to be in the form of a pre-formed
dimer at the higher concentrations of protein required for
binding in the presence of higher salt concentrations.
In Figure 2b, a more striking example of the effect of
lowering the salt concentration on the complex stoichiometry
and also specificity can be seen. The nearly flat profile in
circles corresponds to a titration at 200 mM in KCl of ER onto
a mutant labeled oligonucleotide that exhibits much lower
affinity for the protein than the wild-type sequence (see below).
Squares correspond to ER binding to the wild-type F-vit-ERE in
the presence of 200 mM KCl, and the triangles correspond to
ER binding to the mutant sequence in the absence of added
KCl to the TD buffer. This large increase in anisotropy in the
absence of salt is quite similar to the previously reported
binding of ER to a wild-type rhodamine labeled vit-ERE in the
absence of salt (16), and data on GR-DBD–GRE interactions
under the same conditions (J.J.Hill, C.A.Royer and

2498 Nucleic Acids Research, 2000, Vol. 28, No. 13
K.R.Yamamoto, unpublished results). The very large increase
in anisotropy indicates formation of non-specific complexes of
very high stoichiometry, but reasonably high apparent affinity
since binding begins just above 10 nM in ER.
Effect of ligands on ER–ERE interactions
Although Gorski and co-workers (3,5,16) have reported
previously that ER does not require ligation by estradiol (E2)
for high affinity binding to its response element, others have
reported that cooperative binding depends upon ligation (12),
that tamoxifen liganded receptor releases partially its
tamoxifen ligand upon binding to EREs (12) and that binding
sensograms using SPR are highly ligand dependent (13). Such
behavior would implicate energetic coupling between DNA
binding, ligand binding and even protein–protein interactions.
These observations of specific ligand effects on interaction
with DNA may arise from the use of non-equilibrium assay
techniques, unpurified receptor, in vivo assays and complex
response elements. Besides our recently reported anisotropy
assays (16), no in vitro studies had been carried out under true
equilibrium conditions using purified receptor, and the quality
of our recently reported data lacked somewhat in precision.
Moreover, we did not test ER ligands other than estradiol (E2)
in that study. In order to clarify whether or not ligands play a
role in modulating ER–ERE interactions, we have carried out
the equilibrium anisotropy-based titrations in the absence and
presence of a variety of ER ligands.
Figure 3a and b shows the binding profiles of ER to the inter-
nally labeled fluorescent vit-ERE in the absence of ligands and
in the presence of estradiol (E2) (Fig. 3a) or the partial agonist
4-hydroxytamoxifen (OH-Tam) or the antagonist (ICI) (Fig. 3b)
at 200 mM KCl and 21°C in TD buffer. The results of the analysis
of the data in Figure 3a and b can be found in Table 1. The
reader may remark on a difference in apparent affinity between
the profiles observed in absence of ligand in Figure 3a and b.
This is due to slight differences in the DNA binding activity
between each lot of receptor. We note that active receptor
concentration is determined by the supplier using a radio-
labeled E2 binding assay. However, we have noted these small
differences between batches of ER and, therefore, when
comparing the effects of various assay conditions we are
careful to make these comparisons using the same batch.
Regardless of the batch or the type of ligand used, the apparent
affinity of ER for the ERE does not change significantly with
ligand.
Figure 3. Ligand dependence ER–ERE interactions. (a) F-vitERE titrated
with ERα in the absence of ligand (squares) and in the presence of 0.1 mM E2
(circles); (b) F-vitERE titrated with ERα in the absence of ligand (squares), in
the presence of 0.1 mM ICI (triangles) and in the presence of 0.1 mM OH-Tam
(circles). The profiles were obtained in TD buffer in the presence of 200 mM
KCl at 21°C, and the DNA concentration was 1 nM. In this case the DNA target
was labeled through a fluorescein labeled thymine residue at position +5 from
the 5′-end. Lines through the points represent fits to the data in terms of the
model described in the Materials and Methods. The points plotted at the lowest
protein concentration were obtained in absence of protein.
Effect of temperature on ER–ERE interactions
protein at this temperature. We note that full-length ER is quite
fragile and we have had to take great care in the titrations to
minimize mixing and heating in order to obtain reproducible
results. In any case, the effects of temperature are very small,
indicating that the enthalpy change upon binding is relatively
small, and that the binding process is entropy driven. These
results are consistent with a very in-depth study of Lundback
and co-workers on the thermodynamics of the interactions
between GR-DBD and a variety of mutants with target GREs
(14,15).
The binding profiles for ER with F-vit-ERE at 4, 21 and 32°C
are shown in Figure 4. Due to differences in anisotropy at the
different temperatures arising from viscosity effects we have
fit the raw data and then normalized both data and fit for
comparison. It can be seen that there is no significant differ-
ence between these profiles. The values for the recovered free
energies are given in Table 1. Because the differences are not
significant we have not attempted to extract values for the
enthalpy, entropy or heat capacity changes that may accompany
this interaction. There could be a slight increase in apparent
affinity between 4 and 21°C, but then the lack of further
increase between 21 and 32°C could arise from non-linearity in
the temperature dependence due to a heat capacity change or
alternatively could be due to the onset of denaturation of the
Sequence determinants of ER–ERE interactions
Most natural EREs do not present the perfect palindromic
repeat of the vitellogenin ERE. In order to assess the relative

Nucleic Acids Research, 2000, Vol. 28, No. 13 2499
Table 1. Free energy values for ER–ERE interactions
Temperature (°C)
[KCl] (mM)
200
Ligand 0.1 mM
–
∆G°1 (kcal/mol)
∆G°2 (kcal/mol)
∆G°coop (kcal/mol)
21
(Fig. 2)
10.6 0.2
21.8 0.1
0.6
(8.0 × 10–9 M)a
21
(Fig. 2)
250
300
200
200
200
200
200
200
200
200
–
10.4 0.1
9.8+0.1/–0.4
<8.6
20.0 0.1
–0.6
–0.6
>2.4
>2.4
>0.6
>0.5
>–0.5
2.4
(3.8 × 10–8 M)a
21
(Fig. 2)
–
18.7 0.3
(1.4 × 10–7M)a
21
(Fig. 3a)
–
21.9 0.1
(7.4 × 10–9 M)a
21
(Fig. 3a)
E2
<8.6
22.1 0.1
(6.3 × 10–9 M)a
21
(Fig. 3b)
–
<11.2
23.0 0.1
(2.9 × 10–9 M)a
21
(Fig. 3b)
OH-Tam
<11.3
23.1 0.1
(2.7 × 10–9 M)a
21
(Fig. 3b)
ICI
–
<11.8
23.1 0.1
(2.7 × 10–9 M)a
21
(Fig. 4)
10.3 1.0
10.1 0.2
11.6 0.2
23.0 0.1
(2.9 × 10–9 M)a
4
–
21.50 0.05
1.3
(Fig. 4)
(3.3 × 10–9 M)a
32
(Fig. 4)
–
23.9 0.05
0.65
(2.7 × 10–9 M)a
aThe values in parenthesis represent an apparent Kd value calculated from the value of the free energy for the binding of the two monomers divided by 2. This
calculation was necessary because in most cases the binding was cooperative and a simple calculation of the Kd from the free energy of binding of the first
monomer is not possible. This apparent Kd value gives the reader an idea of the concentration at which 50% binding occurs.
The concentration of the target oligonucleotides was between 1 and 2 nM. Conditions were otherwise as noted in the Materials and Methods. Uncertainties on the
recovered free energy values were obtained by rigorous confidence limit testing, which involves refitting the curve at each tested value of the parameter. This
method provides much more realistic (and larger) confidence limits than typically reported based on the diagonal of the correlation matrix. Moreover, with our
method, the correlation between parameters is taken into account, whereas this is not the case otherwise.
contributions of each of the base pairs in the target site to the
ER–ERE affinity we have made systematic symmetric
purine→pyrimidine or pyrimidine→ purine substitutions by
simply inverting the base pair between sense and anti-sense
strands in both half sites for each of the six base pairs of the
half site. The sequences of the mutated targets are given in
Figure 5 and the binding profiles can be found in Figure 6. All
of the mutants exhibited significantly lower affinities than the
F-vitERE target, as can be seen by the large shift to higher
protein concentrations of the binding profiles. We note that
because of the prohibitive cost and limiting concentration of
the ER we could not carry out the titrations above 100 nM in
protein, and thus the binding profiles for the mutated
sequences are far from complete. In order to obtain a semi-
quantitative assessment of the relative affinities of the different
targets, we have made the assumption that the value of the
upper plateau of the anisotropy is the same as for the wild-type
sequence. We have fit the profiles in terms of a simple binding
model, since not enough data could be obtained to treat these
profiles in terms of their eventual binding cooperativity. In
order to compare the affinity for these sequences with that of
the F-vitERE target, we have reported an apparent Kd for the
wild-type target which is obtained using the value for half of
∆G°2 (Table 2). This value can be thought of as an apparent C1/2
value.
Table 2. Effect of base pair mutations on ERα binding to the ERE target
Sequence
mutA mutB mutC mutD mutE mutF CathD
25 42 36 34 81 31 17
Fold loss Kapp
Profiles were obtained using 1–2 nM fluorescein labeled target oligonucleotide
in buffer, pH and 21°C. The Kapp for the wild-type F-vitERE was calculated
using exp[(½ ∆G°2)/RT], and the Kapp for the mutant sequences was estimated as
described in the text. The apparent Kd for the wild type was 8.0 × 10–9 M.
In Figure 6 we can distinguish clear differences between the
raw data of the mutant target profiles, and these differences are
likewise apparent in their apparent Ka values obtained from the
fits (Table 2). The losses in affinity compared to the F-vitERE
target range from 25-fold for the mutA target to 81-fold for the
mutE target. The effect of the mutations can be best understood
by referring to the schematic diagram (Fig. 7) of the contacts

2500 Nucleic Acids Research, 2000, Vol. 28, No. 13
Figure 6. Sequence dependence of ER–ERE interactions. Titrations are shown
for F-vitERE (closed squares), mutA (open triangles), mutB (open circles),
mutC (closed triangles), mutD (diamonds), mutE (asterisks), mutF (open
squares) and CathD (closed circles). Titrations were carried out using 1–2 nM
target DNA in TD buffer at 21°C in the presence of 200 mM KCl. Lines
through the points represent fits to the data for the mutant targets using a simple
binding model, and for the F-vitERE using the cooperative model described in
the Materials and Methods. The point plotted at the lowest protein concentration
was obtained in the absence of protein.
Figure 4. Temperature dependence of ER–ERE interactions. Normalized
titrations for 4 (triangles), 21 (squares) and 32°C (circles). Profiles were
obtained using the 5′-fluorescein labeled F-vitERE at a concentration of 1 nM
in TD buffer with 200 mM KCl. The data were fit and then the raw data and the
fits were normalized for comparison, to eliminate differences due to temperature
dependence of solution viscosity. Lines represent fits of the data to the model
described in the Materials and Methods. The points plotted at the lowest protein
concentration were obtained in the absence of protein.
between the ER-DBD and its target ERE reproduced based on
that given by Schwabe and co-workers (20) and based on their
crystallographically determined structure. That mutations to
the outermost base pairs of the half-site (mutA, 25-fold and
mutF, 31-fold) result in the smallest perturbations to the
affinity is understandable since no direct contacts to these
bases are apparent in the crystal structure. Only phosphate
contacts appear to extend to these bases. It is in fact somewhat
surprising that the observed effect is so large, corresponding to
~1 kcal/mol per half-site. It is also surprising that the TA→AT
mutation of the outermost of the central base pairs of the half-
site (mutD), which is one of the two base pairs that determines
the ER–GR specificity (22), resulted in only a 34-fold loss in
apparent affinity, even though the thymine at that position
appears to make contact with lysine 32 of ER-DBD (20).
However, since this residue makes a number of other contacts
with the target, there may be some compensatory mechanism.
Mutation (GC→CG, mutC) of the other central base pair that
determines the GR–ER specificity results in only a slightly
larger loss in affinity (41-fold), even though lysine 32 and
glutamate 25 make a number of direct and water-mediated
contacts to the 06 and N7 of the guanine, and the N4 of the
cytosine. In fact, the loss in affinity upon mutation of the
second base pair in the half site (mutB, GC→CG) perturbs the
interaction to the same extent (42-fold). In this case, lysine 28
makes a contact to the O6 of the guanine. The largest perturbation
is found for the CG→GC mutation (mutE) of base pair at posi-
tion 5 of the 1-AGGTCA-6 half site. At this position arginine
33 of the ER-DBD appears to make contacts with both the N7
and the O6 of the guanine, although the contact to the O6
appears to be a water-mediated bifurcated contact involving
Figure 5. Sequences of the wild-type and mutant oligonucleotides used in the
present study. These targets were all 5′-labeled with fluorescein through a six-
carbon phosphoramidite linker as described in the text.

Nucleic Acids Research, 2000, Vol. 28, No. 13 2501
coupled energetically to DNA binding. We have found a very
strong effect of salt concentration on the affinity and the
specificity of ER–ERE interactions. This observation is important,
not simply from a structural point of view, but also to underline
the importance of comparing the results of studies carried out
under similar conditions. We find that below 200 mM in KCl,
we begin to see the appearance of higher order non-specific
complexes. At very low salt the ER exhibits very little, if any,
specificity and very high stoichiometry. Between 200 and
300 mM KCl, the salt concentration dependence of the overall
affinity is fairly consistent with the number of phosphate
contacts observed in the crystallographically determined
structure of the complex between the ER-DBD and its palin-
dromic target (20). The temperature studies revealed almost no
effect of changing temperature on the ER–ERE affinity. Of
course in the absence of any large effect we cannot hope to
analyze the interactions for the thermodynamic parameters
associated with the binding, but these studies do indicate that
the enthalpy of interaction must be rather small, and thus that
the binding is entropy driven. This is consistent with studies of
the GR-DBD–GRE interactions reported earlier (14,15). We
also demonstrate unambiguously the lack of any effect of
ligand binding (agonist or antagonist) on the equilibrium
binding affinity between the ER and its DNA target at 21°C in
200 mM salt.
Finally, our exploration of the contribution of each of the
base pairs in the palindromic ERE target demonstrates that
each base pair contributes significantly to the overall affinity,
although the ER/GR specificity arises from the identity of the
central two base pairs of the half site. In their analysis of their
crystallographically determined structure, Schwabe and co-
workers (20) conclude that the discrimination of half-site
sequence is not simply a matter of slotting a different discrim-
inatory amino acid into a common framework, but also involves
considerable rearrangement of side-chains to compensate for
changes in local DNA structure. The energetic data provided
here support the idea of a globally determined half-site, in
which the energetic contributions to the overall structure and
affinity are rather evenly distributed throughout the site.
Figure 7. Schematic representation of the interactions between the ER-DBD and
the target ERE reproduced from the schematic given by Schwabe and co-workers
(20) based on their crystallographically determined structure. In our schematic the
half site bases are labeled from 1 to 6 (sense strand) and –1 to –6 (anti-sense
strand). Water molecules are represented by filled circles and H-bonds by arrows.
the O4 of the neighboring thymine. It is perhaps due to this
complex network of interactions that mutation at this position
leads to such a significant loss in affinity. Interestingly, the
target oligonucleotide derived from the sequence of the
cathepsin D ERE (Fig. 5) which exhibits a much larger number of
mutations, particularly in the 5′-half site, yields a significantly
higher affinity than any of the symmetrically mutated targets.
The cathepsin D sequence is only 17-fold less effective than
the F-vitERE target. However, comparing the mutations in the
cathepsin D target (with respect to the vitellogenin-based ERE)
we see that three of the mutations are at the least important
positions (1 and 6) and moreover are A→G (purine→purine)
rather than purine→pyrimidine mutations. Two of the
mutations occur in the central three base pairs between half-
sites and the T→ C mutation at position 4 of the 5′ half site
maintains at least the N7 water-mediated contact with
glutamate 25.
ACKNOWLEDEGMENTS
The authors thank Dr Vincent Cavaillès for helpful suggestions.
This work was supported by the CNRS, INSERM, La Fondation
pour la Recherche Médicale, L’Association pour la Recherche
sur le Cancer and the Région Languedoc-Roussillon. N.P. is a
post-doctoral fellow (INSERM poste d’acceuil recherche
clinique) and Emmanuel Margeat is supported by a doctoral
grant from the French Ministère de l’Education, de la
Recherche et de la Technologie.
CONCLUSIONS
REFERENCES
We have determined the affinity of the specific interaction of
baculovirally expressed human ERα for its target ERE under a
variety of conditions. First of all, the binding is clearly cooperative.
Assuming that the final stoichiometry of the specific complex
is two monomers per target ERE, this implies that the full-
length ER dimer dissociates into monomers upon dilution in
the nanomolar concentration range. The observed cooperativity of
binding definitively rules out simple monomer interaction of
ER with its target and implicates a protein–protein interaction
1. Mangelsdorf,D., Thummel,C. and Beato,M. (1995) Cell, 83, 835–839.
2. McKenna,N.J., Lanz,R.B. and O’Malley,B.W. (1999) Endocrin. Rev., 20,
321–344.
3. Murdoch,F.E., Meier,D.A., Furlow,J.D., Grunwald,K.A.A. and Gorski,J.
(1990) Biochemistry, 29, 8377–8385.
4. Murdoch,F.E., Grunwald,K.A.A. and Gorski,J. (1991) Biochemistry, 30,
10838–10844.
5. Furlow,J.D., Murdoch,F.E. and Gorski,J. (1993) J. Biol. Chem., 268,
12519–12525.

2502 Nucleic Acids Research, 2000, Vol. 28, No. 13
6. Murdoch,F.E., Byrne,L.M., Ariazi,E.A., Furlow,J.D., Meier,D.A. and
Gorski,J. (1995) Biochemistry, 34, 9144–9150.
7. Anderson,I., Bartley,C.R., Lerch,R.A., Gray,W.G.N., Friesen,P.D. and
Gorski,J. (1998) Biochemistry, 37, 17287–17298.
8. Klinge,C.M., Peale,F.V.,Jr, Hilf,R., Bambara,R.A. and Zain,S. (1992)
Cancer Res., 52, 1073–1081.
9. Nardulli,A.M., Greene,G.L. and Shapiro,D.J. (1993) Mol. Endocrinol., 7,
331–340.
10. Obourne,J.D., Koszewsi,N.J. and Notides,A.C. (1993) Biochemistry, 32,
6229–6236.
11. Anolik,J.H., Klinge,C.M., Hilf,R. and Bambara,R.A. (1995)
Biochemistry, 34, 2511–2520.
12. Kuntz,M.A. and Shapiro,D.J. (1997) J. Biol. Chem., 272, 27949–27956.
13. Cheskis,B.J., Karathanasis,S. and Lyttle,C.R. (1997) J. Biol. Chem., 272,
11384–11391.
14. Lundback,T., Cairns,C., Gustafsson,J.-A., Carlstedt-Duke,J. and Hard,T.
(1993) Biochemistry, 32, 5074–5082.
15. Lundback,T., Zilliacus,J., Gustafsson,J.-A., Carlstedt-Duke,J. and
Hard,T. (1994) Biochemistry, 33, 5955–5965.
16. Ozers,M.S., Hill,J.J., Ervin,K., Wood,J.R., Nardulli,A.M., Royer,C.A.
and Gorski,J. (1997) J. Biol. Chem., 272, 30405–30411.
17. Royer,C.A., Smith,W.R. and Beechem,J.M. (1991) Anal. Biochem., 192,
287–294.
18. Royer,C.A. and Beechem,J.M. (1992) Methods Enzymol., 210, 481–505.
19. Royer,C.A. (1993) Anal. Biochem., 210, 91–97.
20. Schwabe,J.W.R., Chapman,L., Finch,J.T. and Rhodes,D. (1993) Cell, 75,
567–578.
21. Record,M.T.,Jr, de Haseth,P.L. and Lohman,T.M. (1977) Biochemistry,
16, 4791–4796.
22. Lefstin,J.A., Thomas,J.R. and Yamamoto,K.R. (1994) Genes Dev., 8,
2842–2856.