Department of Chemistry University of Illinois Urbana, Illinois 61801
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ABSTRACT |
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INTRODUCTION |
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X-ray structures of nuclear hormone receptor LBD complexes with various ligands show that the ligand is fully enveloped by several elements of secondary structure in the lower portion of this domain (2, 3, 4, 5). Notable as well is the fact that ligands with agonist vs. antagonist activity stabilize different conformations, particularly the orientation of the C-terminal helix 12. These alternate helix 12 positions regulate the shape and accessibility of a hydrophobic groove formed by elements of helices 3, 4, 5, and 12, which forms a docking site for the LXXLL sequence motif of the nuclear receptor interaction box (NR box) found in various coregulator proteins (6, 7, 8, 9).
What is not evident from these LBD-ligand structures is the process by which the ligand is able to enter and exit the ligand-binding pocket. The fact that the ligand is completely enveloped by the protein indicates that for ligand to exit this pocket there must be a significant conformational reorganization of a substantial region of the lower portion of the LBD. This is consistent with the very slow rate of ligand dissociation from these complexes, which can be many hours at 04 C and several minutes at 25 C (10, 11).
Conformational reorganization is also thought to be required for ligand
to enter the LBD. For example, in a crystal structure of an LBD without
a ligand (an apo-LBD), that of the retinoid X receptor- (RXR
),
the ligand-binding pocket is shown to be "collapsed" when compared
with that typical for LBD-ligand complexes (12). The conformational
change required to open up this pocket to allow a ligand to enter is
again consistent with the very slow rate of ligand association to
nuclear hormone receptor LBDs. These rates are typically several orders
of magnitude slower (ca. 104
M-1sec-1)
(10) than typical enzyme-inhibitor association rates, which are
generally close to diffusion limited (108
M-1sec-1)
(13, 14).
In this report we describe experiments in which we have examined the
conformation of the LBD of the estrogen receptor (ER
) during
progressive chemically induced denaturation with guanidine
hydrochloride (Gua-HCl) using fluorescence methods. Whether we follow
the Gua-HCl-induced unfolding titration of this domain using intrinsic
tryptophan fluorescence or bis-anilinonaphthalenesulfonate (bis-ANS), a
probe for accessible interior regions of proteins, we find evidence
that the ER
-LBD undergoes a two-phase unfolding process. The first
phase (at low Gua-HCl) involves partial unfolding of the LBD, which
opens the ligand-binding pocket, providing access to water, which
quenches the tryptophan fluorescence, and allowing bis-ANS to bind. The
second phase (at high Gua-HCl) corresponds to the global unfolding of
the whole domain, at which point the tryptophans become fully exposed
to water and experience a red shift in their fluorescence. The
denaturant-induced state of the ER
-LBD that is partially unfolded
may resemble a conformation that this domain accesses transiently under
native conditions, allowing ligands to enter or exit the ligand-binding
pocket.
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RESULTS |
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The LBDs of the ERs contain three tryptophan residues (W365, 388, and
398 in ER and W312, 335, and 345 in ERß) that are at congruent
positions on helices 3 and 5 of the two ER subtypes. The location of
these three tryptophan residues within the tertiary structure of the
ER
LBD is illustrated in the ribbon diagram shown in Fig. 1
. As is evident from this figure, all
three of these tryptophans are in the interior of the LBD; they are
also close to one another (1.312.00 nm), and they are positioned
above the ligand-binding pocket, relatively close to the position where
the ligand is bound (0.851.73 nm) (2). We have found that the
intrinsic fluorescence of the three tryptophan residues in the
ER
-LBD provides very useful information on the state of quencher
access and solvation of the internal environment of the ER-LBD during
the process of chemically induced denaturation.
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The tryptophan emission spectra of the ER-LBD in high Gua-HCl (>
4.5 M) solutions agreed well with the fluorescence spectra
of an equimolar aqueous solution of N-acetyl tryptophan,
which was found to have a maximum fluorescence intensity centered about
356 nm (not shown). This indicates that in high Gua-HCl, the
tryptophans of ER
-LBD are fully solvated by water.
Tetrahydrochrysene methyl ketone (THC-ketone) is a fluorescent ligand
we developed for ER that has been used in a variety of studies on ER
ligand binding kinetics, competitive binding assays, and visualization
of ER in cells by fluorescence microscopy (21, 22, 23). It has a good
binding affinity for ER (RBA of 68%, vs. 100% for
estradiol), and in previous work, we have shown that THC-ketone is an
effective quencher of the intrinsic tryptophan fluorescence when it is
bound to the receptor (11). As shown in Fig. 3, THC-ketone binding to ER
-LBD
results in a 90% decrease in Imax relative
to that of the empty receptor. This quenching of intrinsic tryptophan
fluorescence by ER-bound THC-ketone is greater than that of water. In
fact, the level of fluorescence from ER
-LBD with THC-ketone bound is
considerably less than that of an equimolar solution of
N-acetyl-tryptophan in water (corrected for the three
tryptophans in ER
-LBD). As the Gua-HCl concentration is raised on
the ER
-LBD-THC-ketone complex, ligand is released from the receptor
ligand binding pocket at approximately 23 M, at
which point the tryptophan Imax increases
to a level equivalent to that of ER
-LBD in both the unliganded and
estradiol-liganded experiments (cf. Fig 2
, A and B). The THC-ketone has
no effect on the second phase of denaturation (red shift in
em,max at 4.5 M, not
shown).
We also performed analogous experiments with ERß-LBD (either
unliganded or liganded with estradiol or THC-ketone) and found that
this ER subtype showed two-phase denaturation profiles that were nearly
identical to those with ER-LBD (data not shown).
The Nonspecific Fluorescent Probe bis-ANS Suggests That ER-LBD
Denaturation Proceeds through a Partially Folded Intermediate
bis-ANS is a compound that fluoresces strongly in hydrophobic,
nonaqueous environments, but is almost nonfluorescent in water. Because
of these characteristics, it has proved to be a very useful probe of
accessible protein interiors (24, 25). In particular, it is considered
to be a "detector" of protein molten globule states,
i.e. a "liquid-like" state in which the protein has a
largely condensed structure with most of the secondary and some of the
tertiary structure formed, but in a much more dynamic or fluid-like
state than a fully folded protein (26). Because it has essentially no
binding affinity for the native ER (RBA = 0.00031% for bis-ANS),
bis-ANS functions effectively as a probe of the "accessibility" of
hydrophobic regions of the ER-LBD, which operationally can be
considered to be its molten globule character (24, 25).
bis-ANS probing of the denaturation profile of the ER and ERß-LBDs
(both unliganded and estradiol-liganded) was conducted after
equilibration with increasing concentrations of Gua-HCl, in the
presence or absence of estradiol. Figure 4A
shows that with ER
-LBD in the
absence of estradiol, bis-ANS fluorescence intensity
(Imax) reaches a maximum at approximately
0.5 M Gua-HCl and begins to fall at higher
concentrations. In contrast, with ER
-LBD in the presence of
estradiol (Fig. 4
), bis-ANS fluorescence requires approximately 2
M Gua-HCl to reach maximum intensity. Again, with
ER
-LBD liganded with the lower affinity ligand, EED, an intermediate
level of Gua-HCl (ca. 1.5 M) is required for
bis-ANS fluorescence to reach a maximum (not shown). Figure 4B
shows
that ERß-LBD in the absence of estradiol has a peak
Imax at approximately 1
M Gua-HCl, which is shifted to approximately 2
M Gua-HCl when estradiol is present.
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DISCUSSION |
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A reasonable interpretation of our experimental results is that
at low denaturant concentrations, the LBD undergoes a partial
unfolding, reaching a state that allows some water molecules to enter
the interior of this domain, which brings them close enough to the
three interior tryptophans to quench their emission significantly (Fig. 5, state B). However, the tryptophans do not experience a wavelength
shift because the unfolding is only partial, and thus they remain in a
"protein-like" interior. The partially unfolded state also
provides an accessible hydrophobic protein interior phase into which
bis-ANS can enter and exhibit its characteristic enhanced emission.
However, this state appears no longer able to bind ligands stably, as
is evident from the loss of ligand-tryptophan resonance energy transfer
quenching by the THC-ketone ligand. The further unfolding of the LBD
that occurs at much higher concentration of denaturant finally exposes
the tryptophans to the high dielectric of an aqueous medium, causing
the expected red shift in their emission (Fig. 5
, state C). During this
final unfolding, the accessible hydrophobic protein interior character
is also lost, resulting in reduced fluorescence from bis-ANS.
One nuclear receptor LBD crystal structure is very relevant to the
partially unfolded conformation that we are proposing for the ER-LBD.
The structure of the unliganded peroxisome proliferator-activated
receptor (PPAR
) shows that the ligand-binding pocket, which
constitutes the "lower half" of this domain, is considerably more
expanded than it is in a ligand-receptor complex (9, 29), whereas the
structurally conserved "upper half" of this domain has the tightly
folded, compact conformation that is typical for ligand-receptor
complexes. The expanded conformation of the ligand binding pocket of
apo-RXR
is reminiscent of the sorts of partially unfolded structures
that are proposed for protein molten globules (see below), and it may
be similar to the partially unfolded conformation that we are proposing
is induced in ER-LBD by treatment with low concentrations of
denaturant.
Other apoproteins in which cofactors, such as hemes, have been removed, are used as experimental systems to study protein molten globules. Molten globules are conformational states having most of the secondary and some of the tertiary structure of the native protein, but are more dynamic or fluid-like, and they are often proposed as intermediates in protein folding or unfolding. Good examples of these dynamic, partially unfolded states are apomyoglobin at pH 4 (30, 31) and apoflavodoxin in Gua-HCl (18, 19, 32). The finding that certain other apoproteins can be induced to adopt a molten globule state upon treatment with denaturants (or a pH shift) is reminiscent of our finding that the unliganded ER-LBD, also an apoprotein, can be induced to adopt such a partially unfolded, dynamic state upon Gua-HCl treatment.
Ligand Binding Confers Structural Stability to the ER-LBD, as
Evidenced by Its Protective Effect against Guanidine Denaturation
Higher concentrations of Gua-HCl are required to effect a decrease
in the tryptophan fluorescence or an increase bis-ANS fluorescence in
ligand-bound ER-LBD than in unliganded receptor. This demonstrates that
ligand binding does indeed provide structural stability to the receptor
(Fig. 5, States A, B, and C). Ligand binding is protective only toward
the initial phase of denaturation, i.e. partial unfolding;
it does not retard the onset of the red shift in the tryptophan
fluorescence, i.e. global unfolding.
Ligand-induced conformational stabilization is not unique to the
ER-LBD. In the PPAR example noted above (9, 29), ligand binding
causes the open apo-PPAR
structure to collapse to a tightly folded
conformation that is typical of nuclear hormone LBD-ligand
complexes (12). More recently, Johnson et al. (33) have used
nuclear magnetic resonance to document this ligand-induced
stabilization of the ligand binding pocket in PPAR
, and related work
in ER has been done by Luck et al. (34). Pissios et
al. (35) has described another approach to study
ligand-induced changes in the ER LBD. When the LBD is covalently cut
between helix-1 and the rest of the LBD, these two elements interact
weakly, if at all, in the absence of ligand, but they do interact when
estradiol is added. This suggests that ligand binding stabilizes the
packing of helix-1 against the remaining portions of the LBD to such a
degree that it remains associated by strictly noncovalent interactions.
Since helix-1 interacts only with the upper zone of the LBD, ligand
binding apparently has a stabilizing effect on the conformation of this
upper zone.
Our recent finding that the binding of coactivator peptides to the ER-LBD dramatically slows the rate of ligand dissociation is also supportive of the competition between those forces that stabilize the tightly folded conformation of ER and those unfolding motions that are required for ligand dissociation (11). By binding in the coactivator groove, these peptides appear to lock the ER-LBD in the folded conformation, effectively trapping the ligand in the binding pocket to an even greater degree.
These observations and others also highlight differences between in vitro experiments and the cellular context. In all of the experiments described above, only the LBDs of these receptors are being studied alone and in vitro, whereas in the cell, these receptors are full length, which may have some effect on ligand binding (10), and there are, as well, many other proteins with which these receptors can interact. The latter include chaperone proteins that bind preferentially to unliganded receptors and may stabilize them in an unfolded state (36, 37). In fact, with the glucocorticoid receptor, association of the aporeceptor with chaperone proteins appears to be essential for ligand binding (38, 39). Thus, the results from our experiments on the ER-LBD in vitro may not directly predict the behavior of full-length ER in vivo. Nevertheless, they are instructive of the conformational dynamics that are an inherent property of the ER-LBD.
The Dynamic Character of the ER-LBD Is Essential for Its Regulation
of Transcription
All of our results suggest that the LBD is a dynamic protein
capable of entering multiple conformational states (Fig. 5): it needs
to be dynamic for ligand to enter and exit; in the absence of ligand,
it needs to be in a less rigid or soft state that does not afford
effective docking sites for coactivator proteins (state A), and in the
presence of ligands, it needs to adopt stable conformations that
provide the sort of rigid exterior texture that is required to promote
the interaction of coactivators (state D). This general need for the
ligand to induce conformational stability is inherent to the ability of
the ligand to turn on or turn off the activity of the receptor, a
situation that is now widely appreciated in regulatory proteins (40, 41).
Normally, ER is not exposed to denaturants that can induce the partial
unfolding of this domain that would facilitate ligand binding and
dissociation. Yet, both apo-ER and ligand-bound ER must be able to
access such a state under native conditions, although they would need
to do so only transiently (Fig. 5, state B). On
the basis of the studies we have performed here, we propose that ER
under native conditions can transiently access a partially unfolded
intermediate in which the ligand-binding pocket has a more open,
dynamic character, allowing ligand binding, dissociation, and exchange
to occur freely. Whereas such a transient, dynamic state would not be
detectable under native conditions, it appears that an intermediate
that displays the character expected from this partially unfolded state
can be induced in the ER-LBD at intermediate concentrations of
Gua-HCl.
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MATERIALS AND METHODS |
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ER Preparations
The human ER-LBD, comprising amino acids 304554 of human ER
(hER), was expressed from a pET-15b vector in BL21(DE3)pLysS E.
coli and purified as described previously (10). A pET-15b ERß
construct (ERß-LBD) coding for the corresponding amino acids in ERß
(256505) (42) was also prepared using the same cloning site, and the
ERß-LBD was expressed and purified in the same fashion as the
ER
-LBD.
Tryptophan Fluorescence-Monitored Guanidine Denaturation
Assay
For the fluorescence-monitored Gua-HCl denaturation assays of
wild-type hER-LBD, purified hER-LBD was diluted to 1,500 nM
in Tris-glycerol buffer (50 mM Tris, pH 8.0, 10%
glycerol). For assays investigating the effect of ligand binding, the
receptor was saturated by adding E2 or THC to a
final concentration of 4,500 nM. The resultant solution was
then incubated on ice for 1 h before denaturing with the Gua-HCl
stock solutions. A 20 µl portion of the hER-LBD solution was added to
980 µl of a stock Gua-HCl solution, yielding a final hER-LBD
concentration of 30 nM and a final ligand concentration of
90 nM (if ligand was used for the experiment). The Gua-HCl
concentration of each stock solution was determined using the method
described by Pace and Scholtz (43). The hER-LBD/Gua-HCl solutions were
then further incubated on ice for 1 h. An aliquot (900 µl) of
the solution was placed into a 5 x 5 mm quartz fluorescence
cuvette, and the cuvette was placed into the sample chamber of a
Fluorolog II (model IIIc) fluorometer (Spex Industries, Inc., Edison,
NJ). The sample chamber was held at a constant 4 C, and the sample was
excited at 285 nm (1.25-mm slits), and the tryptophan emission was
observed from 300 nm to 420 nm (1.25-mm slits). Five emission scans
were averaged for each sample, and a blank spectrum (buffer alone) was
subtracted and was smoothed using a 21-point Savitsky-Golay algorithm
using the Datamax software (Spex Industries, Inc.). The emission
spectra were then analyzed for peak emission intensity and wavelength
using Excel 97 (Microsoft Corp., Redmond, WA).
A 90 nM solution of N-acetyl tryptophan in Tris-glycerol buffer was used to determine the fluorescence spectral profile of fully solvated tryptophan. This spectrum was determined in an identical fashion to that of hER-LBD, and it showed an emission maximum at 356 nm.
bis-ANS Probing of Receptor Conformational Flexibility
For these experiments, hER-LBD was denatured in Gua-HCl
solutions that had been prepared as described above. All samples were
prepared as in the tryptophan fluorescence-monitored denaturation assay
except that purified hER-LBD was initially diluted to 2,000
nM and the ligand concentrations were adjusted accordingly.
To denature the protein, 15 µl of the 2,000 nM hER-LBD
solutions were diluted into 980 µl of the stock Gua-HCl solutions to
achieve a final hER-LBD concentration of 30 nM. A 5 µl
portion of 0.2 M bis-ANS in EtOH was then added to each
sample to yield a final bis-ANS concentration of 1 mM, and
the samples were further incubated on ice for 1 h. The
fluorescence of bis-ANS was measured exactly as for the tryptophan
fluorescence except that the samples were excited at 294 nm and the
emission spectra were scanned from 430 to 600 nm. The collected data
were analyzed using the same methods as described above, but the
wavelength of maximum emission intensity was not determined.
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FOOTNOTES |
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This research was supported by NIH Grant PHS 1R37 DK-15556.
Received for publication July 28, 2000. Revision received September 25, 2000. Accepted for publication October 13, 2000.
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REFERENCES |
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