(Received for publication, January 30, 1995; and in revised form, August 2, 1995)
From the
Retinoic acid exerts its many biological effects by interaction
with a nuclear protein, the retinoic acid receptor (RAR). The details
of this interaction are unknown due mainly to the lack of sufficient
quantities of pure functional receptor protein for biochemical and
structural studies. We have recently subcloned the D and E domains of
human RAR for expression in Escherichia coli. Using
nickel-chelation affinity chromatography with a polyhistidine
amino-terminal tail, purification of the DE peptide with a pI of 5.18
was accomplished to greater than 98% purity. Scatchard analysis and
fluorescence quenching techniques using the purified protein indicate a
very high percentage of functional molecules (>95%) with a K
for retinoic acid (t-RA) of
0.6 ± 0.1 nM. Circular dichroism spectra of the
purified domains predict a predominantly
-helical structure
(
56%) with little
sheet present. No significant changes in
these structural characteristics were observed upon binding of t-RA. Inspection of the amino acid sequence within these
domains identified a single tryptophan residue at position 227.
Modeling the amino acid sequence in this region as an
-helical
structure indicates that this tryptophan is adjacent to alanine 234,
which corresponds to alanine 225 in RAR
that has previously been
linked to the ligand binding site. Fluorescence of this tryptophan was
quenched in a dose-dependent manner on the addition of t-RA,
confirming that Trp-227 is within the ligand binding site. Tryptophan
flourescence quenching analysis also demonstrates that a single
retinoic acid molecule is bound per receptor and suggests that
receptor-ligand interactions occur within the amino-terminal portion of
the predominantly
-helical ligand binding domain.
Retinoids, derivatives of vitamin A, play important roles in
morphogenesis, differentiation, and cellular
proliferation(1, 2, 3) . Their action, at the
molecular level, is mediated by several nuclear receptors belonging to
the steroid/thyroid receptor superfamily(4) . Three retinoic
acid receptors (RAR, (
)-
, and -
) bind
all-trans retinoic acid (t-RA) and 9-cis RA,
while three retinoid X receptors (RXR
, -
, and -
) bind
9-cis RA but not t-RA(5, 6, 7) . As a class, these
receptors are ligand-inducible trans-acting transcription
factors, which can modulate the expression of specific target genes by
interaction with cis-acting DNA sequences termed retinoic acid
response elements(8, 9, 10) .
The RARs, like other members of this nuclear receptor superfamily, have a modular structure consisting of six domains denoted A through F (11, 12, 13) . The E region, or ligand binding domain, is 85-90% conserved among the RARs and has ligand-dependent transactivation and dimerization functions(14, 15, 16) .
Information
pertaining to the interaction between the RARs and their ligand
requires large quantities of purified receptor protein in a functional
three-dimensional conformation. Several attempts have been made at
purification of recombinant full-length RAR, -
, and -
expressed in eukaryotic cells(6, 17, 19) ,
Sf9 insect cells using the baculovirus expression
system(18, 19) , and a variety of expression systems
in Escherichia coli(20, 21) . Expression of
subfragments corresponding to the RAR ligand binding domain (i.e. domains DEF, EF, etc.) in E. coli improved yields as
compared to expression of full-length
receptors(17, 22, 23) . However, in every
case, regardless of yield, the purified receptor preparations were
insufficiently active with respect to ligand binding and were therefore
unsuitable for structural or biochemical analysis.
Improved
expression and purification techniques described here using nickel
chelation chromatography and a modified thrombin cleavage produced
milligram quantities of hRAR receptor protein containing the D and
E domains. This DE
protein was found to have binding kinetics
similar to that of the native full-length receptor. Circular dichroism
analysis of the expressed protein suggests that the secondary structure
of the ligand binding domain is predominantly
-helical and
contains very little
sheet. This observation is in good agreement
with the reported crystal structure of the ligand binding domain of
RXR
, a closely related member of the steroid/thyroid hormone
receptor superfamily(24) , and that determined from structure
prediction modeling. Interestingly, the relative pattern of secondary
structural components for this peptide did not change significantly
upon binding to ligand.
Inspection of the amino acid sequence within
the D and E domains revealed a single tryptophan residue at position
227, which was subsequently used for fluorimetric titration. At a
concentration of protein determined by amino acid analysis,
fluorescence was quenched in a dose-dependent manner upon addition of t-RA with a transition point equal to the concentration of
protein in the reaction. Cogan analysis (33) of this curve
indicates a single ligand binding site within DE. Further, the
position of the transition point indicates that >95% of the receptor
protein is present in an active conformation and that Trp-227 is
located in close proximity to the ligand binding site. The assignment
of Trp-227 within the ligand binding site is further supported by a
previous report that Ala-234 (Ala-225 in RAR
) is a contact amino
acid within the ligand binding site(25) . In an
-helical
structure, this alanine would be expected to be immediately adjacent to
Trp-227 supporting a structural model for the ligand binding pocket.
For denaturation studies, flourescence measurements
were obtained by diluting the purified DE protein in 6 M
guanidine HCl, and emission spectra were obtained as described above in
the absence of t-RA.
Figure 1:
SDS-polyacrylamide gel electrophoresis
analysis of DE purification. Samples from the purification of
DE
were separated on a 12% SDS-polyacrylamide gel and silver
stained. Lane 1, crude soluble extract. Lane 2,
unbound protein. Lanes 3 and 4, 60 mM imidazole washes. Lane 5, thrombin-cleaved DE
. Lane 6, 400 mM imidazole fraction. Closed
arrowhead indicates position of HIS
-DE
; open
arrowhead indicates position of
DE
.
The HIS-DE
peptide was purified from the crude soluble cytoplasmic extract by
nickel chelation chromatography. The yield was approximately 7 mg of
DE
from 383 mg of crude protein (Table 1) representing about
2% of the total soluble protein and is reported as the average of five
experiments. The HIS
-DE
fusion is undetectable in the
void volume (Fig. 1, lane 2), indicating that the
expressed fusion protein was efficiently bound to the affinity resin.
Repeated washing of the affinity column with 60 volumes of a 5 mM imidazole buffer followed by 40 volumes of a 60 mM imidazole buffer was necessary to completely remove contaminating E. coli proteins from the resin.
The purified DE
protein was eluted by thrombin cleavage of the resin-bound
HIS
-DE
fusion. Based on quantitative estimates using
laser densitometry, greater than 85% of the HIS
-DE
peptide was cleaved by thrombin. Approximately 60% of the cleaved
product was recovered in the column eluate, and the purity of this
protein was greater than 98% (Fig. 1, lane 5). Results
from amino acid analysis of the protein in this fraction indicate that
the composition of residues in the purified DE
protein is
consistent with that determined from the primary sequence
so that the protein preparation is unlikely to be contaminated.
Subsequent treatment of the column with buffer containing 400 mM imidazole elutes the remaining DE
protein (Fig. 1, lane 6). Since the DE
in this fraction does not bind
retinoic acid (data not shown) and spontaneously precipitates upon
collection, it most likely contains molecules that are incorrectly
folded.
The isoelectric point (pI) for the purified DE protein
was determined on a Pharmacia IEF 3-9 Phast Gel and was found to
be 5.2. This experimentally obtained pI value is in good agreement with
that calculated from the primary amino acid sequence (5.18) (Fig. 2).
Figure 2:
Determination of the isoelectric point of
human E. coli-derived DE protein. Broad pI calibration
standards were run on a Pharmacia IEF 3-9 Phast Gel and silver
stained. Isoelectric points of the standards are plotted against their
migration distance from the cathode (pI points are listed in parentheses): lentil lectin basic (8.65), lentil lectin middle
(8.45), lentil lectin acidic (8.15), horse myoglobin acidic (6.85),
human carbonic anhydrase B (6.55), bovine carbonic anhydrase B (5.85),
-lactoglobulin A (5.20), soybean trypsin inhibitor (4.55), and
amyloglucosidase (3.50). The open square indicates the
position of purified DE
protein.
Figure 3:
Saturation binding and Scatchard analysis. a, 1.0 pmol of purified DE was incubated with the
indicated concentrations of [
H]t-RA in
the presence or absence of a 100-fold molar excess of unlabeled
retinoic acid as described under ``Experimental Procedures.''
, total binding activity;
, specific binding activity;
, nonspecific binding activity. b, Scatchard plot of the
binding data. The calculated K
was 0.60
nM (±0.1), where n =
3.
Figure 4:
Structural analysis of purified DE
protein expressed in E. coli. A, secondary structure
prediction of amino acids 155-421 of DE
after Garnier et
al.(34) . B, CD spectra (plotted as the molar
ellipticity, [
], versus wavelength) are shown
for DE
protein in the absence (line b) and presence (line a) of 1 µMt-RA.
The
tryptophan fluorescence emission spectrum was used to evaluate the
integrity of the three-dimensional structure of the folded DE
protein. The fluorescence of the native protein exhibits two maxima at
320 and 340 nm (Fig. 5). Upon denaturation with 6 M guanidine HCl, Trp-227 fluorescence shifts to higher wavelengths
(340-350 nm), indicating increased exposure of that residue to
the solvent. This shift is most likely the result of a repositioning of
the tryptophan residue into a more polar, less shielded environment and
is an indication that the purified DE
protein was properly folded
prior to denaturation. At the same time, relative fluorescence maxima
of both the native and denatured protein remained the same (68 versus 75%), consistent with the view that fluorescence
quenching was not the result of changes in conformation.
Figure 5:
Fluorescence emission spectra of denatured
DE protein. A shift in the emission maxima from 340 to 350 nm is
observed upon denaturation of DE
protein with 6 M guanidine HCl.
Trp-227
fluorescence can also be used to monitor binding kinetics of the
purified receptor protein. Titration of 5 µM DE
protein with increasing concentrations of t-RA reveals
significant quenching of the 340-nm fluorescence peak (Fig. 6a). Since the concentration of t-RA at
the inflection point of the curve (5.1 µM) is very close
to the concentration of receptor protein in each reaction vessel (5.0
µM determined by amino acid analysis),
greater
than 95% of receptor molecules are folded into a functionally active
conformation. Further, linearization of the fluorescence quench data by
the method of Cogan (33) (Fig. 6b) was used to
demonstrate the presence of one ligand binding site per receptor
molecule (n = 1.08) from the slope of the Cogan curve.
Figure 6:
Fluorimetric titration of purified
DE. a, intrinsic fluorescence of DE
protein is
quenched upon addition of increasing amounts of t-RA.
Fluorescence was measured at 340 nm emission after excitation at 280 nm
using a slit width of 5 nm. b, titration quench data are
linearized to determine the number of binding sites per receptor
molecule (n) after the method of Cogan et
al.(33) . Po = protein concentration; Ro = retinoid concentration;
= (F - F
/Fo - F
) where Fo = initial
fluorescence, F = fluorescence at each Ro, and F
= fluorescence at maximum
quench.
Information concerning the three-dimensional structure of members of the steroid/thyroid hormone receptor superfamily has been hindered by the inability to purify sufficient quantities of receptor protein for NMR or x-ray crystallographic analysis. Several laboratories have reported on the use of expression systems in different species as a method for increasing yields of heterologous protein, but the yields of purified protein with functional activity have generally been low(6, 17, 18, 19, 20, 21, 22, 23) . Another approach has been to express each domain separately under the assumption that a three-dimensional structure obtained in this way would be identical to that of the same domain in the context of the full-length receptor protein. The most successful use of this approach has been the expression and resulting NMR analysis of the DNA binding domain of the glucocorticoid(35, 36) , estrogen(37, 38) , retinoic acid(39) , and retinoid X (40) receptors. The structure of the DNA binding domain has rendered it particularly amenable to functional expression in both prokaryotic and eukaryotic systems due mainly to the hydrophilicity of the amino acids in this zinc finger domain. Other domains, such as the ligand binding domain E, have proven more difficult to produce in high yields due to the large number of hydrophobic residues in this region.
The method described here
consistently produced 5-8 mg of purified DE protein from 1
liter of bacterial culture, representing approximately 30% recovery of
expressed protein (Table 1). Purity and integrity of protein
obtained in this way was assessed by a number of criteria.
Silver-stained polyacrylamide gel electrophoresis gave a single band of
the expected molecular mass, 31,000 daltons (Fig. 1, lane
5). The experimentally determined pI of the purified DE
protein (Fig. 2) agrees with that calculated from the amino acid
sequence (5.2 versus 5.18). Binding and Scatchard analysis of
the purified protein gave a K
of 0.6 nM (Fig. 3). This value is consistent with that obtained using
crude protein extracts(7) . In addition, competition binding
assays using other naturally occurring retinoids (Table 2) gave
apparent K
values similar to those published for
the full-length receptor (17, 20) with the same order
of affinity: t-RA > 9-cis RA > retinol >
retinal.
Since the DE protein appeared to be purified to
homogeneity by the methods described using the above criteria, it was
appropriate to evaluate the secondary structure characteristics using
circular dichroism. Initial analysis revealed that light scattering of
the sample hindered interpretation of the CD spectra at wavelengths
below 190 nm. Previous studies have shown that truncating the CD
spectrum at 190 nm results in poor estimates for the amount of
sheet and turn conformations found in the
protein(44, 45) . However, the same studies found that
because the CD for an
-helix dominates the spectrum, the analysis
for
-helix is reliable regardless of the wavelength range. Given
these considerations, the purified DE
protein was found to contain
a high degree of
-helical structure. Constrained deconvolution of
the CD spectra to quantitate helix, sheet, or random coil conformations
suggests an
-helical content as high as 56%, in good agreement
with a value of 62%
-helical content determined using the Garnier
algorithm (Fig. 4A). Further support for this
observation comes from the recently published crystal structure of a
closely related member of the steroid/thyroid superfamily, RXR
(24) . This structure contains approximately 65%
-helix
with only 4%
sheet.
On addition of retinoic acid, only minor
changes in helical content were observed (Fig. 4b),
indicating that major alterations in the secondary structure of DE
were unlikely to occur upon ligand binding. Recently, CD analysis of
the thyroid hormone receptor, another member of the steroid/thyroid
hormone receptor superfamily, indicated a high proportion of
-helical structure(46) . Interestingly, addition of
thyroid hormone produced only minor changes in the CD spectra obtained,
in agreement with the results reported here.
The finding of
significant helical content for DE is noteworthy in light of the
fact that the structure of another protein known to bind retinoids,
retinol-binding protein, is primarily
sheet. Retinol binding
protein is a member of a family of proteins whose architecture consists
of a
barrel formed by two orthogonal
sheets and four turns
of
-helix at its carboxyl terminus(47, 48) .
Based on the results of this study, DE
is structurally distinct
from the retinol binding protein family. In support of this
observation, one-dimensional NMR spectra of DE
did not show any of
the typical markers for
sheet conformation. (
)Few
-proton resonances are observed down field of water, and there is
little chemical shift dispersion among the amide proton resonances that
are typical of proteins with significant
sheet or turn
conformations.
Inspection of the amino acid sequence within the
DE protein revealed a tryptophan residue at position 227. This
residue was used to evaluate the native structure of DE
as well as
the kinetics of ligand binding. Denaturation of the protein with 6 M guanidine causes a shift in the emission maxima from 340 to
350 nm (Fig. 5). This shift suggests a repositioning of Trp-227
into a more polar environment(41) . Since the relative
fluorescence does not change, this shift is most likely due to
structural differences common upon denaturation but not a change in
autofluorescence. Thus, any quenching observed upon the addition of
ligand will be due to ligand binding only, further supporting the
position of Trp-227 within the ligand binding site.
Upon addition of
increasing t-RA concentration, the stoichiometry of ligand
binding to purified DE protein can be measured. This method has
been used to evaluate the binding of retinoic acid and other naturally
occurring retinoids to the cytoplasmic retinoic acid binding
proteins(41, 42) . Fig. 6a shows that
the fluorescence of Trp-227 is quenched in a dose-dependent manner. An
inflection in this binding curve can be expected at the concentration
of ligand, which saturates all the binding sites present within the
protein tested. As can be seen in Fig. 6a, fluorescence
is progressively quenched upon addition of increasing amounts of t-RA. The inflection point of this titration curve occurs at a
point slightly greater than 5 µMt-RA. Since the
concentration of DE
protein in each reaction vessel was adjusted
to 5 µM based on amino acid analysis, it is evident that
stoichiometric binding of a single binding site occurs for greater than
95% of the purified protein molecules. Linearization of the
fluorimetric titration data for DE
using the method of Cogan et al.(33) confirms only one t-RA binding
site per receptor molecule (n = 1.08) (Fig. 6b). This result appears to be a reliable
estimate of the number of binding sites (n) since this method
is most accurate when high affinity binding occurs, as is the case for
DE
(43) .
The results presented here predict that the
ligand binding pocket of RAR will be composed of at least one
-helical structure containing the tryptophan at position 227. A
model of this region (Fig. 7) shows the location of Trp-227.
Interestingly, this residue is immediately adjacent to an alanine
residue at position 232. This residue is analogous to Ala-225 in
RAR
, which has been recently demonstrated to be an important
recognition residue for receptor-selective retinoids(25) .
Maksymowych et al.(49) have predicted a similar
helix-turn-helix motif in this region for all members of the
steroid/thyroid hormone receptor superfamily. Taken together, these
results suggest that the region in the amino-terminal portion of the
ligand binding domain of the RARs contains the ligand recognition site
and that all the members of this superfamily are likely to contain
receptor-ligand contact points in this region. In addition, modeling
the position of 9-cis RA within the ligand binding domain of
another member of this superfamily, RXR
, from the crystal
structure (24) predicts receptor-ligand contacts within the
hydrophobic portion of the homologous helix that is described here for
RAR
, further supporting this region as the ligand binding site.
Figure 7: A helical model for the amino-terminal portion of the receptor ligand binding domain. The secondary structure of the ligand binding domain for amino acids 210-272 is represented by a two-dimensional helical net diagram. Trp-227 and Ala-234 are highlighted. Hydrophilic residues are marked by dark circles. Hydrophobic residues are in the open circles.
In summary, this work describes an efficient, one-step method for
the production and purification of milligram quantities of human
RAR DE domains expressed as heterologous protein in E.
coli. The purified DE
receptor protein is of high purity and
significant biological activity. The protein exhibits binding
affinities consistent with the full-length receptor for t-RA
and other naturally occurring ligands. Secondary structural analysis
using circular dichroism is consistent with a predominantly
-helical polypeptide with only minor alterations in secondary
structure evident upon ligand binding. Fluorescence quenching
techniques were used to identify a single hydrophobic,
-helical
site within the amino-terminal portion of the ligand binding domain,
which contains a tryptophan residue in the ligand binding pocket.
Future studies will assess the structural and biochemical properties of
this binding site.