The Conformation of the Glucocorticoid Receptor AF1/tau1 Domain
Induced by Osmolyte Binds Co-regulatory Proteins*
Raj
Kumar,
J. Ching
Lee,
D. Wayne
Bolen, and
E. Brad
Thompson
From the Department of Human Biological Chemistry and Genetics,
University of Texas Medical Branch, Galveston, Texas 77555
Received for publication, January 29, 2001, and in revised form, March 8, 2001
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ABSTRACT |
The activation domains of many transcription
factors appear to exist naturally in an unfolded or only partially
folded state. This seems to be the case for AF1/tau1, the major
transactivation domain of the human glucocorticoid receptor. We show
here that in buffers containing the natural osmolyte trimethylamine
N-oxide (TMAO), recombinant AF1 folds into more a compact
structure, as evidenced by altered fluorescence emission, circular
dichroism spectra, and ultracentrifugal analysis. This conformational
transition is cooperative, a characteristic of proteins folding to
natural structures. The structure resulting from incubation in TMAO
causes the peptide to resist proteolysis by trypsin, chymotrypsin,
endoproteinase Arg-C and endoproteinase Gluc-C. Ultracentrifugation
studies indicate that AF1/tau1 exists as a monomer in aqueous solution
and that the presence of TMAO does not lead to oligomerization or
aggregation. It has been suggested that recombinant AF1 binds both the
ubiquitous coactivator CBP and the TATA box-binding protein, TBP.
Interactions with both of these are greatly enhanced in the presence of
TMAO. Co-immunoadsorption experiments indicate that in TMAO each of these and the coactivator SRC-1 are found complexed with AF1. These
data indicate that TMAO induces a conformation in AF1/tau1 that is
important for its interaction with certain co-regulatory proteins.
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INTRODUCTION |
Transcriptional activation by the glucocorticoid receptor
(GR)1 is mediated through the
function of three regions in the protein, AF1/tau1, tau2, and AF2.
Numerous mutational analyses have shown that of these, AF1/tau1 makes
by far the largest quantitative contribution to transcriptional
activation. Deletion or inactivating mutations of AF1/tau1 reduce the
ability of the GR to activate transcription from test genes by at least
60-70% (1-3). As defined by molecular genetic analyses, tau2 and AF2
are located in the C-terminal ligand-binding domain (LBD) of the
receptor, whereas AF1/tau1 lies N-terminal from the centrally located
DNA-binding domain (DBD). In the human GR (hGR) AF1 is encompassed by
amino acids 77-262 (3). Precisely how the AF1/tau1 transactivation region functions is unknown, in part due to lack of knowledge of its
working structure; yet this structure must provide the basis for
essential, specific interactions with other proteins. Studies
undertaken in our laboratory and others (4, 5) have shown that when
expressed independently in dilute aqueous solutions, AF1 appears to
have little structure. Instead it appears to exist as a collection of
conformers that overall appears as random coil. However, in the
presence of the strong
-helix stabilizing agent trifluoroethanol, as
many as three segments toward the C-terminal end of the AF1/tau1 region
exhibit
-helical characteristics (4). Mutations in the GR tau1/AF1
transactivation region coupled with assays of function have suggested
that this ability to form
-helical conformation in vitro
correlates with the transactivation potential of the region (4). The
subdomain of AF1/tau1 that subsumes the potential helices has been
referred to as the AF1 core (AF1c), and when this core is
expressed separately while connected to an exogenous DNA-binding
domain, AF1 retains considerable transactivating activity. Mutations of
acidic amino acids in AF1, including the core, had little effect on
such activity; however, mutations altering several hydrophobic residues
of AF1/tau1 significantly diminished its transcriptional activation
potential (6).
In recent years, a number of proteins that modulate GR activity, such
as GRIP1, RIP140, SRC-1, and CBP/p300, have been identified (7-9).
These co-regulators bind the GR and presumably act as molecular bridges
to the primary transcriptional machinery. Most co-regulators have been
identified through their ligand-dependent interactions with
the LBD, in particular, with the AF2 domain in members of the receptor
family of which the GR is part (10-14). However, it is not known
whether the co-regulators function exclusively through this part of the
GR or whether they can also modulate the activity of AF1/tau1. Some may
well do so. CBP and several other co-regulators have been shown to
bind, if not strongly, to recombinant AF1/tau1 (15-17). A recent study
using AF1c or the potential helices thereof connected to a
GR DNA-binding domain could interact in a yeast system with a fragment
of CBP to activate appropriate promoter-reporter constructs (17). AF1
has also been shown to bind TBP (16), the critical TATA box-binding
protein that forms the basis for the multiprotein transcription
initiation complex. This raises the possibility that the AF1 GR domain
somehow directly influences the transcription machinery. In
vitro transcription studies indicated that the holoGR acts to
stabilize the preinitiation complex (18). One possibility is that a
structured conformation of AF1/tau1 is induced or stabilized during its
interaction with specific target proteins. This model in fact has been
supported for the activation domains of some transcription factors (19, 20). Thus, data short of actual structural proof support the idea that
conditional folding of the AF1/tau1 region is an important requirement
for its interaction with target factors and subsequent role in gene
regulation. In vivo, the particular conformer of AF1/tau1
required for transcriptional activation presumably is induced or
stabilized by forces coming from intra- and inter-molecular interactions. Knowledge of those factors and the conformation adopted
by AF1/tau1 will lead to understanding of the role of this region in
the transcription process, information essential to understanding how
glucocorticoids affect gene regulation.
Naturally occurring solutes or organic osmolytes have been used to
shift the thermodynamic balance so as to make unstructured proteins
fold into native-like, functioning structures (21-26). One such
osmolyte, trimethylamine N-oxide (TMAO), has been used very
successfully to fold unstructured, inactive proteins into proteins with
significant functional activity (27, 28). We recently demonstrated that
TMAO induces secondary and tertiary structure in a GR fragment
containing the entire N terminus plus the DBD (amino acids 1-500). The
conformational transition of this GR fragment is cooperative in nature
(5), a condition characteristic of proteins folding to their proper
shape. Because most of the structural changes taking place in this GR
fragment appear to be happening in the N-terminal domain, we
hypothesize that TMAO induces an ordered conformation in the AF1/tau1
region, and this conformation is important for its interaction with
target proteins. In this paper, we present studies of the TMAO-induced conformation in the AF1/tau1 region expressed alone. Our data suggest
that TMAO induces secondary and tertiary structure in the AF1/tau1
region, and this induced conformation greatly enhances its interaction
with certain co-regulatory proteins and TBP.
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MATERIALS AND METHODS |
Expression and Purification--
Construction and expression of
AF1/tau1 has been described (5). The bacteria containing the
recombinant vector for GST-AF1/tau1 were induced with
isopropyl-
-D-thiogalactopyranoside (0.5 mM) for 3 h, lysed, and extracted. The bacterial extracts were loaded onto a glutathione-Sepharose column at 4 °C. AF1/tau1 protein was
eluted from the column by thrombin digestion, followed by a Superdex-75
exclusion column as described (5). For GST adsorption experiments, GST
or GST-tagged protein was purified by eluting the bound protein using
50 mM Tris, 500 mM NaCl, 50 mM
reduced glutathione, pH 8.3. Protein purity was analyzed by
SDS-polyacrylamide gel electrophoresis stained with Coomassie Blue
R-250 and was estimated to be greater than 95%.
Fluorescence Emission Spectroscopy--
Fluorescence emission
spectra of purified AF1/tau1 protein in solution were recorded at
various concentrations of TMAO. The spectra were monitored using a Spex
Fluoro Max spectrofluorimeter at excitation wavelengths of 278 or 295 nm as described (5). All measurements were made in 1-cm rectangular
cuvettes thermostated at 22 °C, and all the data were corrected for
the contribution of the solute concentrations. To prevent aggregation
of protein we added proline at a constant molar ratio of TMAO:proline
as 4:1 in all samples containing TMAO.
Circular Dichroism Spectroscopy--
The CD spectra were
recorded at 22 °C on an Aviv 62 spectropolarimeter using a 1.0-cm
quartz cell, with the bandwidth of 1.0 nm and scan step of 0.5 nm. The
spectra were recorded at a protein concentration of 1.0 mg/ml (in the
presence and absence of TMAO) in 10 mM Tris, 10 mM NaCl, 10 mM dithiothreitol, pH 7.9, and were corrected for the contribution of solute concentrations. Each spectrum
shown is a result of five spectra accumulated, averaged, and smoothed.
Limited Proteolytic Digestion--
Sequencing grade trypsin,
chymotrypsin, endoproteinase Gluc-C, and endoproteinase Arg-C were used
for proteolytic digestions. Digestion was carried out with 5 µg of
purified AF1/tau1 in 10 mM Tris, 10 mM NaCl, 2 mM dithiothreitol, pH 7.9. Except for Endo Arg-C, which was
carried out at room temperature for 50 min, all other reactions were
carried out at 4 °C for 20 min. Endo Gluc-C and Endo Arg-C were
added at a protein:enzyme mass ratio of 50:1, whereas trypsin and
chymotrypsin were added at 100:1. Reactions were terminated by adding
SDS loading buffer and then placing the sample tubes in a boiling water
bath. The samples were then run on SDS-PAGE gel and stained by
Coomassie Blue R-250. For protein microsequencing, the proteolytic
reactions were carried out in triplicate in the presence and
absence of 3 M TMAO. After gel electrophoresis, the
proteins were transferred onto PVDF membranes. The largest protected
bands in the samples containing 3 M TMAO were pooled and
sequenced by five cycles of Edman degradation (29).
Sedimentation Velocity--
Sedimentation was carried out with a
Beckman XL-A analytical ultracentrifuge, using a two-channel Epon
centerpiece. Purified AF1/tau1 protein was sedimented in a buffer
containing 10 mM Tris, 200 mM NaCl, 1 mM dithiothreitol, pH 7.9. Rotor speed was 60,000 rpm with
scans taken at 280 nm. The apparent sedimentation coefficient distribution was determined using the time derivative of the
concentration profile (30) as modified by J. Philo (DCCT+, version
1.11). Standard protocols (31) were used to correct apparent
sedimentation coefficient (s*) to 20 °C and water
(s20,w). The s* values were
corrected for viscosity, which was measured for buffer with and without
3 M TMAO, using a viscometer. For sedimentation equilibrium studies the samples were run at 20,000, 25,000, and 30,000 rpm. The
samples were judged to be at equilibrium when successive scans showed
no change in the distribution of protein. Data were analyzed using
nonlinear least squares parameter estimation using the program NONLIN
(32, 33). The buffer conditions were the same as in sedimentation
velocity measurements.
GST-AF1 Adsorption Assay--
Purified GST-AF1 hybrid protein in
a buffer containing 10 mM Tris, 10 mM NaCl, pH
7.9, with or without TMAO was immobilized on glutathione-Sepharose
beads. Nuclear extracts were prepared from HeLa cells and added (1.0 mg/ml) to the GST-AF1 bound to the beads, and the mixture was further
incubated for 2 h. Any unbound protein was washed thoroughly. The
appropriate concentrations of TMAO were kept throughout the assay. To
the washed beads, SDS-PAGE sample buffer was added, and the sample was
boiled for 5 min in a water bath. Each sample was then run on a
SDS-PAGE gel and visualized by Coomassie Blue R-250 staining. To
confirm CBP binding, CBP was constitutively expressed in COS-1 cells
using transient transfection as described (34), and this COS-1 cell
extract was used in place of HeLa nuclear extracts. All other
adsorptions were carried out using HeLa nuclear extracts. After gel
electrophoresis, proteins were transferred onto a PVDF membrane. The
membrane was first blocked by incubation for 2 h at room
temperature in PBS containing 10% nonfat milk proteins and then
incubated at 4 °C with appropriate antibodies (Santa Cruz
Biotechnology) overnight. The immunoreactive bands were developed using
ECL method and visualized by autoradiography.
Immunoadsorption--
Purified AF1/tau1 and HeLa nuclear
proteins were mixed together in a buffer containing 10 mM
Tris, 10 mM NaCl, pH 7.9 (with or without 3 M
TMAO), to a final volume of 1 ml and incubated for 1 h at 4 °C.
5 µl of antibodies (SRC-1, CBP, or TBP; Santa Cruz) and 25 µl of
protein-agarose conjugate were then added and incubated further for
2 h. Pellets were collected by centrifugation and washed
thoroughly. The beads were resuspended in SDS buffer and boiled for 5 min. After SDS-PAGE gel electrophoresis, the proteins were transferred
onto a PVDF membrane and immunoblotted using an antibody raised against
amino acids 150-175 of the human GR as described above.
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RESULTS |
TMAO Causes the hGR AF1/tau1 Transactivation Region to Fold into a
Native-like Conformation--
As with other related receptors, the GR
contains several major functional domains. These are shown
diagrammatically for the human GR in Fig.
1. The AF1/tau1 transactivation region,
amino acids 77-262 is highlighted, with vertical
lines above the bar indicating the location of the two Tyr and one
Trp residues within AF1/tau1. This domain was expressed in a bacterial
system as a hybrid protein tagged at the N-terminal residue with
glutathione S-transferase (GST). After removing the GST
moiety, we studied the folding of the AF1/tau1 protein. Fluorescence
emission was used to monitor the environment around the Trp and Tyr
residues. The fluorescence emission spectra of >95% pure recombinant
AF1/tau1 are presented in Fig. 2. Fig.
2a shows the spectra in the absence and presence of 3.5 M TMAO after excitation at 278 nm, which reflect the
changes coming both from Tyr and Trp residues. Fig. 2b
indicates the fluorescence emission changes after excitation at 295 nm, which specifically follows changes in the environment of the single Trp
residue located between the first two potential helices of the AF1
core. In both sets of spectra, the quantum yield of the fluorescence is
increased in the presence of TMAO, with an increase of about 2-fold at
3.5 M TMAO. There are blue shifts in the emission maxima in
the presence of TMAO. These fluorescence changes are typical of those
accompanying the removal of aromatic residues from polar, aqueous
solution into a more hydrophobic environment within the protein. Both
the increase in quantum yield and the blue shift in fluorescence maxima
indicate the formation of compact structure in the presence of TMAO.
Because the three amino acids excited are located well apart in
AF1/tau1, the conformational changes reflected in the fluorescence
emission changes may be happening throughout the peptide. TMAO induces
this conformational transition in AF1/tau1 in a cooperative manner, as
shown by monitoring the shift in fluorescence emission maximum after
excitation at 278 nm as a function of TMAO concentration (Fig.
2c). These observations strongly suggest that TMAO folds
AF1/tau1 region into a more compact structure, and the cooperativity of
the process is a hallmark of native protein folding (35, 36).

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Fig. 1.
Topological diagram of human GR showing its
functional regions. The numbers indicate positions of
amino acids. The vertical lines represent the positions of
Trp (bold line), and Tyr (thin lines) residues in
AF1/tau1.
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Fig. 2.
Fluorescence emission spectra of AF1/tau1 in
the absence and presence TMAO. The curves a, b, d and e
show the spectra in the absence (---) and presence (- - -) of 3.5 M TMAO. Experiments a-c were carried out in low
salt; d-f were carried out in high salt. a and
d, excitation at 278 nm; b and e,
excitation at 295 nm. c and f show the reversible
conformational transition of AF1/tau1 induced by TMAO, as monitored by
change in emission maxima upon excitation at 278 nm with respect to
increasing concentrations of TMAO. The spectra in low salt were
recorded in buffer containing 10 mM Tris, 10 mM
NaCl, 10 mM dithiothreitol, pH 7.9. Those of
d-f were recorded in the same buffer except with 200 mM NaCl. The linear least squares best fit of experimental
data to the two-state model of protein folding/denaturation using
linear extrapolation methods (42) gives apparent thermodynamic
parameters of TMAO-induced folding: G = 2.8 ± 1.1 kcal/mol, m = 1.1 ± 0.6 in 10 mM NaCl, and G = 3.9 ± 0.7 kcal/mol, m = 1.6 ± 0.3 in 200 mM NaCl.
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Because the ultracentrifugation studies (discussed below) required a
buffer containing 200 mM NaCl, we repeated the fluorescence emission spectra in this buffer to find out whether the structural changes we observed in AF1/tau1 following TMAO exposure occurred at
this salt concentration. In Fig. 2, (d and e) the
quantum yield of the fluorescence is increased significantly following
TMAO exposure, as was seen in Fig. 2 (a and b).
Interestingly, the blue shift in emission maxima at 295 nm is
significantly increased compared with that at low salt (Fig. 2,
b versus e), indicating that higher
salt concentration favors the conformational transition of folding of
AF1/tau1 by TMAO. The TMAO-induced conformational transition in
AF1/tau1 in 200 mM salt also is cooperative (Fig. 2f) and reaches saturation at 3 M compared with
that of 3.5 M in 10 mM salt (Fig.
2c). These fluorescence emission studies thus indicate that
the TMAO-induced cooperative conformational transition in AF1/tau1 is
more favored at 200 mM.
The fluorescence emission spectra of AF1/tau1 in the presence and
absence of TMAO suggest that TMAO causes tertiary structure to form in
this protein. To acquire further evidence for tertiary structure
occurring in AF1/tau1, we recorded the near-UV CD spectra of this
protein in the presence and absence of TMAO (Fig.
3). Comparison of the spectra shows that
in the presence of TMAO, the maximum at around 290 nm is significantly
increased, reflecting perturbation of the Trp residue. These data
support the conclusion that TMAO causes three-dimensional structure to
occur in the domain.

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Fig. 3.
Near UV-CD spectra of AF1/tau1 in the absence
(---) and presence (- - -) of 3 M TMAO. Each
spectrum is the result of five spectra accumulated, averaged, and
smoothed.
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The Structure Induced in AF1/tau1 by TMAO Resists
Proteolysis--
As another way of evaluating the changes in the
tertiary structure of the AF1/tau1 region brought about by TMAO, we
carried out limited proteolytic digestions of the protein with four
different proteases in the presence and absence of the osmolyte. To be
sure that the compound was not interfering with enzyme activity, we first tested trypsin activity on the artificial substrate,
N
-benzoyl-L-arginine p-nitroanilide. Tryptic activity was not blocked by TMAO. In
fact, proteolysis of the test substrate was somewhat enhanced, perhaps due to the osmolyte preserving the natural active conformation of the
enzyme (data not shown). The patterns of proteolytic products of
AF1/tau1 after digestion by trypsin and chymotrypsin are shown in Fig.
4a. It is evident that the
partial cleavage patterns vary as the concentration of TMAO increases.
At 0 and 0.5 M TMAO, the protein is nearly completely
digested by both proteases (lanes 3 and 4, and
9 and 10), whereas it is partially protected at 1 and 1.5 M TMAO (lanes 5 and 6 and
lanes 11 and 12). At 2 M or higher
TMAO concentrations, AF1/tau1 appears to be mostly protected, suggesting that it has folded into a tertiary structure that
moves the residues attacked by these enzymes to positions not easily reached by them. Similar results were obtained when AF1/tau1 was digested with the proteases Endo Gluc-C and Endo Arg-C (Fig.
4b), although in the case of Endo Gluc-C, the protection was
not as complete. In the case of Endo Arg-C, full protection was seen only at 3 M TMAO.

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Fig. 4.
TMAO-induced folding protects AF1 against
proteolysis by trypsin (lanes 3-8) and chymotrypsin
(lanes 9-14) (a) and endoproteinase
Gluc-C (lanes 2-7) and endoproteinase Arg-C
(lanes 8-13) (b). The lanes
show the products of digestion resolved by SDS-PAGE and stained with
Coomassie Blue. a, lane 1, molecular weight
markers; a, lane 2, and b, lane 1, purified undigested AF1. Each set of 6 lanes shows the results from
digestion in the presence of 0, 0.5, 1.0, 1.5, 2.0, or 3.0 M TMAO.
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We compared the peptide patterns resulting from digestion of
heat-denatured and non-denatured AF1/tau1, to see whether the non-denatured form contained folded regions resistant to peptidases. The data (not shown) indicated no difference in patterns, consistent with lack of significant structure in native recombinant AF1/tau1. We
then compared the peptide patterns of the folded forms induced by TMAO.
Upon exposure to each of the four proteases, both non-denatured and
denatured AF1/tau1 show closely similar protected digestion patterns
after incubation in the presence of 3 M TMAO for 15 min up
to 16 h (data not shown). The similar patterns of protected peptides at every time point in the presence of 3 M TMAO
demonstrate that TMAO can induce similar structures in AF1/tau1 whether
it is initially non-denatured or denatured. Acute (15 min) TMAO
exposure is enough to fold the recombinant protein domain into a
tertiary structure not distinguishable, in this test, from that
produced by the longest exposure. These observations suggest that
once in a sufficient concentration of TMAO, AF1/tau1 folds rapidly to a
protease-resistant shape and that it remains in this conformation, irrespective of the duration of TMAO exposure.
Four of the most prominent protected bands seen after partial digestion
of AF1 in the presence of 3 M TMAO were identified by
sequencing their N termini. In Fig. 5 the
positions of these bands in the AF1/tau1 domain are shown
diagrammatically. A long region starting at the N terminus of AF1/tau1
was protected from digestion by trypsin, chymotrypsin, and Endo Gluc-C.
Many potential substrate sites for these enzymes exist within that
region. All three enzymes cut in a relatively short region lying
approximately beyond amino acid 217. Although we did not identify them
by sequencing as yet, the sizes of smaller peptides protected against
these three enzymes suggest that in TMAO a relatively short segment of
AF1/tau1 beyond amino acid 217 is open to attack. Peptide 4 was
produced by Endo Arg-C, which has only two potential substrate sites in
AF1/tau1. The protected site is Arg-214. This result is
consistent with the interpretation that in the TMAO-folded portion, a
short region beyond amino acid 217 is open to proteolysis. It is
evident from these results that TMAO-induced structure in AF1/tau1 is
not confined to only one part of the molecule.

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Fig. 5.
Location of some major proteolytic fragments
from AF1/tau1, protected by TMAO. Proteolytic digestion data are
combined with protein microsequencing. The horizontal solid
bar diagrams AF1/tau1 (amino acid 77-262). The symbols indicate
the amino acid positions which trypsin, thin vertical line;
chymotrypsin, oval; endoproteinase Gluc-C, thin
black triangle; or endoproteinase Arg-C, wide triangle;
can cut. The horizontal lines indicate the largest protected
peptide after digestion in each enzyme in the presence of 3 M TMAO. Partial sequencing provided identification of the N
termini of these peptides. Sizes estimated by electrophoresis on
denaturing gels.
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AF1/tau1 Exists as a Monomer Both in the Absence and Presence of
TMAO--
It is evident from our data that TMAO exposure apparently
leads to the formation of a significant amount of secondary and tertiary structure in the AF1/tau1. The observed structural changes in
the presence of 3 M TMAO might originate from an
oligomerization of the protein induced by the presence of the
co-solvent. Analytical sedimentation studies were conducted to monitor
the effects of TMAO on the quaternary structure of the protein. Data
from sedimentation velocity studies of AF1/tau1 in the absence and
presence of 3 M TMAO were found to fit a single species
model, as shown in Fig. 6. No additional
species were detected, and the total amount of protein loaded in the
cell was accounted for in that single species. The s*
(s20,w) value for AF1/tau1 obtained in
the presence of 3 M TMAO is
2.4, which corresponds to
the expected value for a globular protein of ~20-25 kDa (37).
However, the s* value for AF1/tau1 in buffer without TMAO is
1.6, consistent with that of a protein of the same molecular weight
but one that assumes either an asymmetric shape or adopts an
unstructured conformation. Sedimentation equilibrium experiments were
also run under similar conditions at three different speeds (20,000, 25,000, and 30,000 rpm). The results (not shown) indicated the presence
of a protein of a molecular mass of 21 kDa, which corresponds to
the monomeric molecular weight for AF1/tau1. Thus these
ultracentrifugation studies clearly indicate that up to 0.5 mg/ml
AF1/tau1 exists as a monomer in the aqueous solutions employed in this
study and that exposure to 3 M TMAO does not induce protein
aggregation.

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Fig. 6.
Sedimentation coefficient distribution of
AF1/tau1 as determined from sedimentation velocity. The peak
s* corresponds to the apparent sedimentation coefficient.
a, in buffer; b, in 3 M TMAO.
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TMAO Facilitates the Interaction of AF1/tau1 with Important Target
Proteins--
It is presumed that AF1/tau1 makes physical interactions
with other factors in order to transactivate gene(s) (16) and that conditional folding is important for these interactions (6). We
therefore evaluated whether the conformation induced in AF1/tau1 by
TMAO is important for specific protein-protein interactions. By
using a GST "pull-down" assay with GST-AF1/tau1 attached to solid
beads as the adsorptive reagent, we observed that in the presence of
TMAO, several proteins from HeLa cell nuclear extracts were adsorbed.
Fig. 7 shows an example of the results.
It is evident from the Coomassie-stained gel that when sufficient TMAO
is present, GST-tau1 binds only certain proteins from HeLa nuclear
extracts. Several higher molecular weight bands that are not prominent
in the extract become so in the adsorbed proteins, and one low
molecular weight band that is prominent in the crude extract is
strongly retained on the GST-AF1/tau1 column. GST alone or GST-AF1 in
low concentrations of TMAO did not retain these proteins. Several similar experiments using both unlabeled and metabolically labeled proteins confirmed the fact that in TMAO certain proteins have high
affinity for AF1/tau1. In the experiment shown, after binding the
proteins to the GST-AF1 on beads in the presence of 3 M
TMAO, the column with bound proteins was washed extensively with a
buffer containing no TMAO. Subsequent elution and
electrophoresis of the retained proteins showed that removal of TMAO
from the washes had not completely released these proteins from the
GST-AF1, suggesting that once the AF1/tau1 complexes were formed in the
presence of TMAO, they were relatively stable (Fig. 7, compare
lanes 5 and 6).

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Fig. 7.
TMAO enhances GST-AF1/tau1
interactions with several proteins from HeLa cell nuclear extract in a
GST pull-down assay. Proteins were identified by SDS-PAGE and
Coomassie Blue staining. Lane 1, molecular weight markers;
lane 2, nuclear extract; lane 3, GST-AF1/tau1
with no nuclear extract; lane 4, GST-AF1/ tau1 + nuclear
extract; lanes 5 and 6, GST-AF1/tau1 + nuclear
extract + 3.0 M TMAO. The beads used in lane 5 were washed with a buffer with 3 M TMAO, whereas in
lane 6, beads were washed with buffer only. Horizontal
lines to the right indicate two of the 5 or 6 proteins
retained in TMAO.
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To begin to identify specific AF1/tau1 target factors, we screened the
bound proteins by immunoreacting these with antisera against a selected
set of known co-regulators. RIP140, CBP, NcoR, TBP, GRIP, p/Cip, SRC-1
have been shown previously to interact with nuclear receptors and/or to
affect receptor-specific transcriptional activation (7-14). No
immunoreactions were observed with the sera we employed for N-CoR,
GRIP1, RIP140, or p/Cip, either in the presence or absence of 3 M TMAO. (One such experiment is shown in Fig.
8.) Consistent with previous
reports (6), in the absence of TMAO we detected a weak interaction with
TBP and CBP. This was increased dramatically in the presence of 3 M TMAO (Fig. 8). In the absence of TMAO the antiserum did
not detect a p250/300 band in the adsorbed HeLa nuclear proteins but
did show a slight reaction with a protein of 160 kDa. But in 3 M TMAO, a reaction at 250/300 kDa and a very strong
reaction at 160 kDa were seen. To determine whether TMAO truly enhanced
an interaction between CBP and AF1/tau1, we transfected COS-1 cells
with a CBP plasmid designed to express CBP constitutively. In extracts
from these cells we found that in the presence of 3 M TMAO
a protein of 250/300 kDa, reactive to the anti-CBP antiserum,
was trapped by the GST-AF1 column (Fig. 8). Among the other HeLa
proteins trapped in the presence of 3 M TMAO on the GST-AF1
column was TBP. Only a weak reaction was seen unless TMAO was
present.

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Fig. 8.
TMAO enhances the association of TBP, CBP,
and SRC-1, but not all known steroid receptor cofactors, with
AF1/tau1. In the upper three panels, GST-AF1/tau1
linked to glutathione-Sepharose beads were used to adsorb proteins from
HeLa cell nuclear extracts (see "Materials and Methods").
Lanes 1, nuclear extract without adsorption to beads.
Lanes 2, adsorption without TMAO. Lanes 3, adsorption in the presence of 3 M TMAO. The data shown are
immunoreactions of the proteins resolved by denaturing gel
electrophoresis, using antibodies to GRIP1, TBP, or CBP. Lower
panel shows the immunoreaction to antibodies against AF1 (resolved
by gel electrophoresis), after immunoprecipitation with the indicated
antibodies. Lanes 1, 3, and 5, immunoadsorption
in the absence of TMAO. Lanes 2, 4, and 6,
immunoadsorption in 3 M TMAO.
|
|
To confirm further these AF1/tau1 interactions, we carried out
immunoadsorption experiments, using CBP and TBP primary antibodies in
HeLa nuclear extracts that had been supplemented with recombinant AF1/tau1. We also probed for adsorbed proteins with an antibody to the
160-kDa protein SRC-1. After allowing the extracts to react with each
antiserum, the protein complexes were trapped by adding secondary
antibodies linked to inert beads. The beads were washed extensively,
and the bound proteins were released and resolved by electrophoresis in
denaturing conditions on polyacrylamide gels. An antiserum to amino
acids 150-175 of the hGR was used to identify AF1/tau1. In the absence
of TMAO, a small amount of AF1 was found to have been retained on the
beads precipitated with anti-CBP and anti-TBP. No SRC-1/AF1 interaction
was seen without TMAO. In the presence of 3 M TMAO, a very
strong interaction of AF1 with each of the other proteins has been
shown to have occurred. Taken together, these protein-protein
interaction data indicate that TMAO-induced folding in AF1/tau1 is
important for its interaction with target factors. Among these may be
CBP, TBP, and SRC-1.
 |
DISCUSSION |
Deletion studies have indicated that hGR lacking the AF1/tau1
transactivation region retains only a small portion of its
transactivation activity (1, 2); hence, it has been concluded that this region plays a major role in gene regulation by GR. It has been reported that AF1/tau1 makes physical contact with certain other proteins to activate genes (6, 15-17). It has also been shown that a
C-terminal portion of AF1/tau1 is indispensable for its transactivation
activity. This "core" region has a propensity to form
-helix,
and in the presence of trifluoroethanol, it can form three helical
segments (4). These potential helices seem important for functional
interactions with a fragment of CBP in a yeast system (17). The induced
fit model of folding hypothesizes that the AF1/tau1 region is
unstructured in vivo until it adopts a more ordered
conformation (38). In this paper we have studied the conditional
folding of recombinant AF1/tau1 in the presence of a naturally
occurring osmolyte, TMAO, which has been shown to fold proteins into
native-like structures (27). Our results clearly demonstrate that TMAO
causes a significant amount of secondary/tertiary structure to form in
the AF1/tau1 region. The fluorescence emission data indicate a shift of
hydrophobic amino acids into a more non-polar environment, just the
type of change seen as natural hydrophobic forces drive protein
folding. In both low salt and near physiological salt concentrations,
the conformational transition is cooperative, a hallmark of naturally
folding proteins (35, 36), and the free energy shift is similar to that
seen during spontaneous folding of globular proteins. Osmolyte-driven
stabilization of protein folding is in fact a process used often in
nature (23). Due to its solvophobic effect on the peptide backbone
(25), TMAO forces thermodynamically unstable proteins to fold to active
conformations without altering the rules for folding (25). Osmolytes,
including TMAO, serve this purpose in a wide range of organisms. Based
on these facts and on our observations, it is highly likely that TMAO
enhances folding to a natural structure in AF1/tau1. The near-UV CD and
the fluorescence emission data both point to this conclusion. These
data monitor signals indicating that the 2 Tyr and 1 Trp residues move
to more hydrophobic locations, e.g. to the interior of the
protein. The TMAO-induced increased s* value without a
change in molecular weight suggests that the protein is more tightly
packed and assumes a globular structure in the presence of this
co-solvent. Since the breadth of the sedimentation profiles shown in
Fig. 6 is proportional to the diffusion coefficient of the protein, the
larger breadth in the profile for AF1/tau1 in the presence of 3 M TMAO indicates a higher diffusion coefficient. A higher
diffusion coefficient for the same molecular weight protein implies
that the hydrodynamic shape of the protein is more compact or
symmetric. Thus, the hydrodynamic data are in complete agreement with
the conclusion that the presence of 3 M TMAO induces
AF1/tau1 to assume a more structured state without inducing formation
of large aggregates. The data from limited protease digestions combined with microsequencing analysis of peptides in the digests indicate that
extensive regions of AF1 are protected, implying that much of the
recombinant peptide is involved.
Based on these observations we hypothesize that an induced conformation
or limited set of conformations occurs in AF1/tau1 in order for it to
carry out its transcription function. This could happen either by an
induced fit mechanism, in which AF1/tau1 would fold as the direct
result of interaction with its target factor(s), or by a shift in the
equilibrium between a large proportion of the unstructured form and a
small proportion of properly structured form (Fig.
9). In the latter case, the small
proportion of structured molecules can make physical interactions with
target molecules. By shifting the equilibrium to favor structured
molecules, TMAO would enhance the interaction of AF1/tau1 with its
partners. Of course, in the holoGR, AF1/tau1 may be partially
structured. Our data on two domain fragments of the GR (39) and the
data of others (40) on the progesterone receptor and its N-terminal region are consistent with this. But no data available suggest even in
the holo-receptor that AF1/tau1 is a fully structured globular
domain.

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Fig. 9.
Model for folding of the AF1 domain of the
GR. AF1U represents the assembly of unfolded
conformers of AF1 in the absence of its proper high affinity protein
binding partner (BP). In this circumstance, AF1 could exist
only in its unfolded state(s) or in equilibrium with a properly folded
state, AF1N. BP could induce folding
directly to AF1N by binding, shifting AF1 directly to the
heterodimer AF1N·BP. Alternatively, AF1U
could be in equilibrium with its native folded state AF1N.
Without BP, [AF1N] is very small relative to
[AF1U]; however, BP could bind AF1N and
eventually shift AF1 to the complex, by the law of mass action. Agents
such as the osmolyte, TMAO, by shifting the equilibrium toward
AF1N, enhance the quantity of AF1 available immediately for
binding to BP.
|
|
If TMAO causes AF1 to assume a native, functional structure, then the
interaction of the domain with partner proteins should be enhanced. It
is very likely that AF1/tau1 makes a physical contact with cofactors or
other proteins to activate gene transcription. It has been shown that
the AF1/tau1 core can bind to several proteins important for
transcription, including CBP and TBP (6, 15-17). These proteins have
also been reported to interact with the LBD of the GR, presumably at
AF2. A careful examination of the data for the observed AF1/tau1
interactions with these proteins shows that although specific, only a
limited amount of protein-protein binding could be demonstrated. This
is consistent with the aspect of our model (Fig. 9) in which a small
portion of AF1/tau1 is in the folded state, capable of such
interactions, whereas most of AF1/tau1 remains unfolded due to the
unfavorable equilibrium of Fig. 9a. Our protein-protein
interaction experiments show that in TMAO, the binding of AF1/tau1 to a
finite number of other cellular proteins is greatly enhanced.
Specifically, the interactions of AF1 with CBP, TBP, and SRC-1 are
enhanced in the presence of TMAO.
Taken together, our observations suggest that TMAO folds the
intrinsically unstructured conformers of AF1/tau1 into a conformation which is well suited for its interaction with other cofactors. Once
AF1/tau1 reaches this proper shape, co-regulator or basal TATA
box-binding protein(s) can interact efficiently with this part of the
receptor and stabilize its structure. We have noticed during our GST
pull-down assays that once the target factors interact, removal of TMAO
does not alter this interaction, suggesting relatively tight binding,
not dependent on constant presence of TMAO. Therefore, it can be
speculated that TMAO thermodynamically favors a well structured
conformation of AF1/tau1, and co-regulator proteins stabilize this
structure. We have also presented data that binding an hGR fragment
containing the DBD to its cognate DNA-binding site causes AF1 to fold
(39). Fig. 10 presents a model in which the available data for AF1/tau1 are accommodated in the context of
holo-receptor. This model leaves open the possibility that additional
interactions occur between the proteins binding at AF1 and other parts
of the GR, e.g. AF2. Some reports suggest that GR AF1 and
AF2 work in conjunction with each other through the DRIP complex (41).
Conformational changes from other factors such as cross-domain
communication, binding to glucocorticoid response elements, or even
steroid binding could also play a significant role.

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Fig. 10.
Model applying Fig. 9 to the GR. The
top schematic shows the GR with its globular DBD and
LBD but a nonspecifically conformed major transactivation domain,
AF1/tau1. The osmolyte TMAO gives structure to AF1 such that specific
binding to co-regulators can occur.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Drs. Alex Kurosky, Bo Xu, and the
staff of the University of Texas Medical Branch Protein Core Laboratory
for their help in overexpressing, purifying, and microsequencing the
AF1/tau1 protein. We also thank Dr. Christopher Chin for
ultracentrifuge studies, Emily W. Welch and Lucy Lee for excellent
technical assistance, and Margie Wronski for help in preparing the
manuscript. The sedimentation study was supported by a Shared
Instrument Grant RR 08961. The CBP construct was kindly provided by Dr.
Tienko, University of Texas Medical Branch.
 |
FOOTNOTES |
*
This work was supported by NCI Grant 5RO1 CA 41407 and
NIDDKD Grant 1R01 DK58829-01 from the National Institutes of Health (to
E. B. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Human Biological Chemistry and Genetics, University of Texas Medical Branch, 301 University Blvd., 605 Basic Science Bldg., Galveston, TX
77555-0645. Tel.: 409-772-2271; Fax: 409-772-5159; E-mail: bthompso@utmb.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M100825200
 |
ABBREVIATIONS |
The abbreviations used are:
GR, glucocorticoid
receptor;
hGR, human GR;
TMAO, trimethylamine N-oxide;
LBD, ligand-binding domain;
DBD, DNA-binding domain;
TBP, TATA box-binding
protein;
GST, glutathione S-transferase;
Endo, endoproteinase;
PAGE, polyacrylamide gel electrophoresis;
PVDF, polyvinylidene difluoride.
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