 |
INTRODUCTION |
The nuclear receptor gene family in humans consists of at least 48 structurally related proteins that regulate transcription of target
genes (1). These include receptors for the steroid and thyroid
hormones, retinoids, vitamin D, prostaglandins, fatty acids, and
unknown ligands, the orphan receptors. Nuclear receptors are comprised
of single polypeptide chains that contain modular domains (2-4). The N
termini of the receptors, which are the most variable in length, have
transcription transactivation functions. The centrally placed and
highly conserved DNA-binding domain
(DBD)1 directs receptor
binding to DNA and is also involved in dimerization. The
carboxyl-terminal ligand-binding domain (LBD) binds the ligand and
undergoes ligand-induced conformational changes that promote dissociation of corepressors and association of coactivators that mediate receptor-induced changes in transcriptional control. The LBD
also participates in receptor homodimerization, heterodimerization, and
oligomerization (5, 6).
Retinoids exert multiple effects on morphogenesis and differentiation
in fetal and adult organs and regulate glucose and lipid homeostasis.
They also act as potent inhibitors of oncogenesis in rodent models and
are used as chemo-preventive and therapeutic agents in several types of
cancers in humans (7-12). In mice retinoid X receptor (RXR) selective
agonists function as insulin sensitizers and can decrease
hyperglycemia, hypertriglyceridemia, and hyperinsulinemia (13). The
three RXR isoforms (
,
, and
) bind to 9-cis retinoic acid and
can function either as homodimers or heterodimers with many other
members of the nuclear receptor family, including the receptors for
thyroid hormones (TRs), vitamin D, and the peroxisome proliferator-activated receptors. RXR homodimers bind preferentially to
direct repeats (DRs) of the AGGTCA half-site spaced by one nucleotide
(DR-1), whereas heterodimers with peroxisome proliferator-activated receptors, vitamin D receptors, TRs, and retinoic acid receptors bind to DRs spaced by 1, 3, 4, or 5 nucleotides, respectively.
The RXR has been reported to exist either as a mixed population of
monomers, dimers, and tetramers (14) or monomers and tetramers (15) in
solution. In the absence of ligand, the values for
Kd monomer
dimer and
Kd dimer
tetramer are 130 and 2.8 nM, respectively, as calculated from fluorescence anisotropy titration studies, indicating that in this setting tetramer
formation is favored (14). Addition of ligand to tetramers in solution
leads to their rapid dissociation to form homodimers (14, 15).
Moreover, unliganded RXR dimers bind DR-1 relatively strongly, but the
tetramers do not. Addition of DR-1 DNA to tetramer-containing solutions
leads to formation of dimers. Thus, both ligand and the RXR cognate DNA
element shift the dynamic equilibrium between RXR tetramer and dimer so
that dimer formation is enhanced.
A large number of isolated nuclear receptor DBD and LBD structures have
been obtained in crystal and in solution. The RXR
DBD has been
solved in complexes with DNA as homodimers and as heterodimers with the
TR DBD and retinoic acid receptor DBD (16-18). The RXR
homodimer DBD binds to the DR-1 element as a head to tail dimer. The
distance between the DR-1 half-site sequences is ~3 nm, and part of
the
-helix connecting DBDs to LBD (T-box) unwinds allowing efficient
DNA binding and DBD dimerization (18). RXR LBDs have been crystallized
in a variety of forms, including dimers and tetramers (19-23). The
LBDs are mostly
-helical with the ligand buried in the interior of
the holo-receptor and contributing to formation of the hydrophobic core
of the protein. There are significant structural differences between
the RXR LBD apo- and holo-forms. In particular, transactivation helix
(H12) protrudes from the body of the receptor in the absence of ligand,
but packs against the body of receptor in its presence (19, 20, 24,
25). This repositioning of H12 completes formation of a hydrophobic cleft that also contains residues of H3, 4, and 5 and forms a docking
site for LXXLL motifs in nuclear receptor
coactivators. The RXR LBD dimer interface overlaps residues from the
intersection of helices 7, 9, and 10 (21) and corresponds to the
position of the TR homo- and heterodimerization interface as defined by
site-directed mutagenesis (26). A crystal structure of an RXR-LBD
tetramer, along with solution x-ray scattering studies of the same
oligomer (21, 22), reveals that it consists of a head to head "dimer of dimers" with a large (2750 Å2) interface comprised of
residues from H3, H11, and H12 (discussed further below). This
extensive tetramer interface is thought to contribute to the marked
stability of this oligomer (27). Comparison of the liganded homodimer
structure with the tetramer structure also suggests a reason for the
observed ligand-dependent dissociation of tetramers to
dimer pairs. Each of the constituent LBDs of the tetramer adopts the
typical apo-receptor conformation, in which H12 protrudes from the body
of the LBD but then, instead of protruding away from the receptor,
actually docks into the H3-H5 hydrophobic cleft region in the adjacent
dimer utilizing its LMEML sequence that resembles the coactivator
LXXLL motif (21). Since ligand repositions H12, addition of
ligand to tetramers would be expected to remove this part of the
interdimer interface and destabilize the tetramer.
While each of these crystal structures has provided major insights into
receptor function, it will be necessary to understand the relationships
between distinct receptor domains to completely understand how nuclear
receptors work. For example, it is not clear why the RXR dimer binds
DR-1 elements with higher affinity than the tetramer. The structures of
any nuclear receptor containing multiple domains have not yet been
solved. In this study, we report the structural organization of a hRXR
protein containing the linked DBD and LBD (hRXR
AB) as revealed by
solution synchrotron x-ray scattering studies. We used the available
crystallographic structures of the isolated hRXR
DBD and LBD domains
to place them inside low resolution ab initio small-angle
x-ray scattering (SAXS) models, which allowed us to assemble models of
both a dimer and a tetramer of hRXR
AB. The results show that the
dimer is a U-shaped molecule with the two DBDs in close proximity (2 nM) forming the tips of the U and separated by an angle of
10°. By contrast, the tetramer is a more elongated X-shaped molecule
formed by two dimers in head to head arrangement in which the DBDs are
extended from the structure and further apart (6 nm) than in the dimer
configuration and are separated by an angle of ~30°. These solution
structures represent the first x-ray structural studies of nuclear
receptors that contain more than one domain and may explain why RXR
dimers recognize target genes with higher affinity than tetramers.
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MATERIALS AND METHODS |
Protein Expression and Purification--
The hRXR
AB
construct encompassing amino acid residues 126-462 was overexpressed
in Escherichia coli cells from strain BL21(DE3) harboring a
pET28a(+) plasmid (Novagen). A Luria broth (LB) starter culture was
inoculated with a single colony of a LB-agar culture and grown
overnight at 37 °C. The initial culture was inoculated at 1% in a
2× YT culture (1.6% tryptone, 1% yeast extract, 0.5% NaCl w/v) and
grown at 37 °C in 50 µg/ml kanamycin medium until A600 nm reached 0.8. Then 5 µM of
zinc sulfate and 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside was added, and the
culture was allowed to grow for 2 h of incubation at 37 °C.
After this cells were harvested by centrifugation, and the pellets were
resuspended in 10 ml/liter culture of buffer A (50 mM
sodium phosphate, pH 7.5, 100 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 10 mM imidazole).
Phenylmethylsulfonyl fluoride and lysozyme were added to 1 mM and 220 µg/ml, respectively, and the culture was placed on ice for 30 min. The lysate was sonicated for six times in 1-min intervals (power 40, Branson Sonifier 450), keeping the culture on ice, and then clarified by centrifugation for 1 h at 20,000 rpm in a Sorval SS34 rotor. The supernatant was loaded onto a
1.5-ml (per liter culture) Talon Superflow Metal Affinity Resin
(Clontech) packed in a c10/10 column (Amersham
Biosciences) and equilibrated in buffer A (flow rate 70 cm/h).
The column was washed with buffer A, until the
A280 nm of the eluent returned to baseline, and
then subsequently with buffer B (50 mM sodium phosphate, pH
7.5, 300 mM NaCl, 10% glycerol, 2 mM
2-mercaptoethanol, and 10 mM imidazole). Then, buffer A was
applied until the conductivity returned to that expected for the buffer
A. The protein was eluted with buffer C (50 mM sodium
phosphate, pH 7.5, 100 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 300 mM imidazole).
Fractions containing hRXR
AB were pooled, diluted 1:1 with a
buffer of 10 mM Tris-HCl, pH 7.5, 10% glycerol, 5 mM DTT (dithiothreitol), and 0.5 mM EDTA. The
fractions were then loaded onto Q-Sepharose High Performance Resin (7 ml, Amersham Biosciences) and packed in a c10/10 column (Amersham
Biosciences) (1 × 10 cm, flow rate 70 cm/h) that had been
pre-equilibrated with this same buffer (plus 50 mM NaCl).
The column was then washed with 10 ml of a buffer of 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10%
glycerol, 5 mM DTT, and 0.5 mM EDTA to remove
unbound protein and eluted with a gradient of 50-500 mM
NaCl. The protein eluted in two peaks, and the fractions of each peak
were pooled and concentrated. Protein content and purity of all
chromatographic fractions were checked by Coomassie Blue-stained SDS
gels. Protein concentrations were determined in parallel using the
Bio-Rad dye assay and bovine serum albumin as standard.
Size Exclusion Chromatography and Dynamic Light Scattering
Experiments--
The protein oligomerization state was assessed by
size exclusion chromatography and dynamic light scattering. Each of two protein elution peaks was separately concentrated and loaded onto a
Superdex 200 size exclusion column (1 × 30 cm, Amersham
Biosciences). The column was pre-equilibrated with a buffer of 10 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 5 mM DTT at a flow rate of 0.5 ml/min and standardized with
the molecular mass calibration kit (Amersham Biosciences) using 100 µl of protein standard samples of a known Stokes radii
(thyroglobulin, 8.5 nm; ferritin, 6.1 nm; catalase, 5.22 nm; aldolase,
4.81 nm; bovine serum albumin, 3.55 nm; ovalbumin, 3.05 nm;
chymotrypsinogen, 2.09 nm, and ribonuclease A, 1.64 nm). The different
oligomeric forms of RXR
AB receptor were analyzed under the same
conditions. The initial protein concentrations of 2.2, 5.6, and 12.3 mg/ml (peak 1) and 3, 8.2, and 11 mg/ml (peak 2) were studied.
Dynamic light scattering measurements were performed with a DynaPro
MS200 instrument (Protein Solutions) at 4 °C using a 12-µl cuvette. The protein samples were concentrated to 1.17 and 7 mg/ml (separately peak 1 and peak 2) in 10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol, and 5 mM DTT prior
to measurements.
Small-angle X-ray Scattering Measurements and Data
Analysis--
SAXS data were collected at the small-angle
scattering beamline on the LNLS (National Synchrotron Light Laboratory,
Campinas, Brazil) using one-dimensional position-sensitive detector
(28). hRXR
at the concentrations 2, 3.1, 6.3, and 14 mg/ml was
measured at a wavelength
= 0.138 nm for sample-detector
distances of 632.5 and 1345.2 mm covering the momentum transfer range
0.1 < q < 6 nm
1 (q = 4
sin
/
, where 2
is the scattering angle). The scattering curves of the protein solutions and the corresponding solvents were
collected in a number of short 30-s to 1-min frames to monitor radiation damage and beam stability. The data were normalized to the
intensity of the incident beam and corrected for detector response.
The scattering of the buffer was subtracted, and the difference curves
were scaled for concentration. The distance distribution functions
p(r) and the radii of gyration Rg were
evaluated by the indirect Fourier transform program GNOM (29). The
molecular masses of the oligomers of hRXR
in solution were estimated
by comparison of the extrapolated forward scattering I(0) with that of
a reference solution of bovine serum albumin with a known molecular mass of 66 kDa. Prior to the shape analysis, a constant was subtracted from the experimental data to ensure that the intensity at higher angles decays as q
4 following Porod's law for the
homogeneous particles (30). The value of the constant is derived
automatically from the outer part of the curve by linear fitting in
coordinates q4I(0) versus q4 by the
shape determination program DAMMIN (31). This procedure reduces the
contribution from scattering due to the internal protein structure and
yields an approximation of the "shape scattering" curve
(i.e. scattering from the excluded volume of particle filled by constant density).
The experimental pair distribution functions do not contain any
negative part, which is an indication of the absence of interference effects in the scattering curves produced by spatial correlations. This
argues that all solutions were sufficiently dilute to remove interference effects. The structure function that describes
interparticle correlations may be equal to unity even at high
concentrations for proteins of very anisotropic shape (32).
Shape Determination--
Low resolution particle shapes were
restored from the experimental SAXS data using two ab initio
procedures. In the first procedure (33, 34), the shape is represented
by an angular envelope function, parameterized in terms of spherical
harmonics using multipole expansion methods (35). The maximum number of the spherical harmonics L is selected to keep the number of
free parameters M = (L + 1)
6 close to the number of Shannon channels Ns = Dmaxqmax/
in the
experimental data (36).
The computed scattering intensity of the envelope is compared with the
experimentally obtained one. The envelope is modified by minimizing the
discrepancy
between the calculated and the experimental data (36,
37). The discrepancy
is defined in Equation 1,
|
(Eq. 1)
|
where N is a number of the experimental points
and Iexp(qj) is
the experimental intensity and
(qj) is its
standard deviation in the jth point.
The shapes of the hRXR
dimers and tetramers were also restored from
the experimental data using another ab initio method as
implemented in DAMMIN (31). A sphere of diameter
Dmax was filled by a regular grid of points
corresponding to a dense hexagonal packing of small spheres (dummy
atoms) of radius r0
Dmax. The structure of the dummy atom model
(DAM) is defined by a configuration X, assigning an index to
each atom corresponding to solvent (0) or solute particle (1). The
method searches for a compact interconnected configuration
X, minimizing the goal function in Equation 2,
|
(Eq. 2)
|
where
> 0 is the weight of the looseness penalty (31).
Starting from the initial spherical configuration, simulated annealing is employed for the minimization (31).
Both models assume that the protein structure in solution can be
described by a constant electron density over its whole volume. However, short range fluctuations in electronic density that exist inside the proteins actually yield a constant contribution at small
q. To apply SASHA and DAMMIN this constant contribution should be previously subtracted from the experimental scattering curves. The assumption of a two-electron density model (corresponding to the protein and the solvent) is progressively weaker for increasing q outside the range covered in the present measurements.
The coordinate sets for both hRXR
LBD and DBD domains were obtained
from Protein Data Bank (PDB accession numbers 1G1U and 2NLL) (16, 19).
Relative positions of the LBD and DBD domains were found by iterative
rotation of their envelope functions to minimize the discrepancy with
the ab initio low resolution structure using an automated
procedure. The models were displayed using the program MASSHA (38).
Radii of gyration (Rg), maximum intraparticle
distances (Dmax), envelop functions, and
scattering curves, were calculated from these atomic coordinates with
use of the program CRYSOL taking into account the influence of the hydration shell (39). SUPCOMB (40) was used to superimpose ab
initio low resolution models with crystallographic structures.
 |
RESULTS |
SAXS Measurements of hRXR
DBD-LBD Dimers and Tetramers--
The
bacterially expressed hRXR
AB preparation eluted from the
Q-Sepharose high performance resin ion exchange column in two peaks (1 and 2; see "Material and Methods"). The protein of both chromatographic fractions migrated on SDS-polyacrylamide gels at a
molecular weight that is consistent with hRXR
AB region at high
purity (>95%; "Material and Methods"). Thus, our preparation of
hRXR
AB exists in two distinct forms. To assess the structural organization of the hRXR
AB in solution, separate synchrotron SAXS
measurements were performed on each peak. Experimental scattering curves from hRXR
are presented in Fig.
1, and the structural parameters derived
from these curves are given in Table
I.

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Fig. 1.
Experimental solution scattering
curves of hRXR AB and results of
the fitting procedures. a, dimer. b,
hRXR AB tetramer. Above, log I versus
q focusing on the fitting of the experimental curve at high
q. Below, details of the same curve at small
q in linear scale with an inset containing the correspondent
Guinier plots (log I versus q2).
(1), experimental curve; (2) experimental curve
after subtraction of a constant value as described under "Materials
and Methods"; (3) scattering intensity from the
DAMs (DAMMIN); (4) scattering intensity from the envelope
model (SASHA).
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|
The hRXR
AB oligomers eluted in peaks 1 and 2 correspond to
molecular masses of 79 and 185 kDa, respectively, as determined in SAXS
experiments by comparison with a reference solution of bovine serum
albumin (Table I). This suggests that peaks 1 and 2 are composed
respectively of hRXR
AB dimers and tetramers. Further support for
this finding comes from the size exclusion chromatography analysis
(Fig. 2). Peak 1 protein Stokes radius was equal to 4.48 and 4.66 nm at protein concentrations of 5.6 and 12.3 mg/ml, respectively. Peak 2 protein Stokes radius varied from 5.2 to
5.7 nm at several protein concentrations between 3 and 11 mg/ml.
Dynamic light scattering experiments conducted on peak 1 and peak 2 fractions at the concentrations 1.2 and 7 mg/ml gave, respectively,
experimental Stokes radii of 4.42 and 4.3 nm for peak 1 and 5.13 and
5.42 nm for peak 2. These results are both consistent with the idea
that peaks 1 and 2 represent dimer and tetramer, respectively.
Determinations of Stokes radius from size exclusion chromatography and
dynamic light scattering agree with Stokes radii calculated from
proposed low resolution SAXS models of the hRXR
AB dimer and
tetramer (see below), which are 4.15 nm for dimer and 5.6 nm for
tetramer (41).

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Fig. 2.
Representative size exclusion
chromatograms of peak 1 and peak 2 protein fractions. RXR AB
from Q-Sepharose peak 1 and peak 2 elution fractions at initial
concentrations of 5.6 and 8.2 mg/ml, respectively, were loaded onto
size exclusion Superdex HR200 10/30 column (Amersham Biosciences).
Respective elution profiles are shown as a dashed line (peak
1) and a solid line (peak 2). The value at the top of each
peak eluted from size exclusion column corresponds to the elution
volume of the RXR AB. The presence of the receptor in these peaks
was confirmed by SDS-electrophoresis (data not shown). The elution
volumes were used to calculate the Kav values
(Kav = (elution volume column void
volume)/(column total volume column void volume)). The value of
was used to determine the
Stokes radius for each peak from the column calibration plot (shown as
an inset). The column calibration plot was obtained eluting
the protein standards of known Stokes radii. Thyroglobulin, ferritin,
catalase, aldolase, albumin, ovalbumin, chymotrypsinogen, and
ribonuclease with the correspondent Stokes radii of 8.5, 6.1, 5.22, 4.81, 3.55, 3.05, 2.09, and 1.64 nm, respectively, were employed.
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|
Concentration of the protein from peak 1 from 3 to 14 mg/ml yielded
SAXS curves that were virtually identical to the SAXS data of the
protein from the elution peak 2 at similar concentration (data not
shown). This suggests that the hRXR
AB dimer population in peak 1 can be converted to a tetramer population at high protein concentration
and is consistent with the previous idea that the DBD-LBD dimer and
tetramer populations are in dynamic equilibrium that is influenced by
protein concentration (14, 15).
The experimental values of Dmax and
Rg, 19 and 5.27 nm for the tetramer and 11.5 and
3.38 nm for the dimer, respectively, suggest that the protein in both
peaks is rather elongated (Table I). The profiles of the distance
distribution function p(r) in Fig. 3 are
typical for elongated particles (42). Nevertheless, the
Dmax of the tetramer (19 nm) is 7.5 nm larger
than the dimer (11.5 nm), which indicates that the tetramer is more
elongated than the dimer.

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Fig. 3.
Distance distribution functions of the
hRXR AB dimer and tetramer.
Distance distribution functions of hRXR AB dimer and tetramer are
given in hollow circles and filled circles,
respectively.
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Ab Initio Shape Determination Using Envelope Functions
(SASHA)--
Shape determinations of the hRXR
dimer and the
hRXR
tetramer were performed using multipole expansion
methods. The experimental data were fitted ab initio by the
scattering from an envelope function starting from a spherical initial
approximation (33, 34). The envelopes were represented with
spherical harmonics up to L = 4 (13 independent
parameters) and L = 10 (31 independent parameters) for
dimer and tetramer, respectively. This was justified by the fact that
the portions of the scattering curves used for ab initio
shape determination using the envelope functions contained Ns = 6.7 Shannon channels for hRXR
dimer and
Ns = 14.1 Shannon channels for hRXR
tetramer. The P2 symmetry was imposed in the case of dimer and P222 in
the case of tetramer.
The restored envelope for the dimer is displayed in Fig.
4, and the fits to experimental data
presented in Fig. 1. The crystallographic models of hRXR
LBD
homodimer and two hRXR
DBD fragments could be unambiguously
positioned inside the envelope for the hRXR
dimer. The restored
envelope for the tetramer is displayed in Fig.
5, and fits to the experimental data are
displayed in Fig. 1. By contrast to the dimer, one hRXR
LBD tetramer
and four hRXR
DBD fragments fit an envelope for the hRXR
tetramer. Two models of the hRXR
LBD dimers obtained by
superposition of the crystallographic structures with ab
initio hRXR
dimer structure were placed within the tetramer
ab initio models allowing for their relative rotation and
translation around common 2-fold axis and adjustment of a relative
orientation of the DBDs. This superposition fits the ab
initio retrieved low resolution tetramer structure. Stokes radii
calculated on the basis of retrieved low resolution structures of dimer
and tetramer is equal to 4.15 and 5.6 nm, respectively (41). These
values agree with experimental values observed for dimer and tetramer
populations in size exclusion chromatography and dynamic light
scattering.

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Fig. 4.
SAXS
hRXR AB dimer envelopes derived
from ab initio calculations. a, upper
row shows a superposition of the envelop model computed by SASHA
(34) to the average of ten DAMs obtained by DAMMIN (31), and the
lower row represent the superposition of the same ten DAMs
to the x-ray structure of the hRXR LBD dimer (21) and two hRXR
DBDs (23). Grid space is 2 nm. b and c are the
same model rotated counterclockwise by 90° around the z-
and y-axes, respectively.
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Fig. 5.
SAXS
hRXR AB tetramer models obtained
by two independent ab initio approaches.
a, upper row shows a superposition of the envelop
model obtained by SASHA (34) to the average of ten DAM calculated by
DAMMIN (31), and the lower row represents the superposition
of the same ten DAM to the x-ray structure of the hRXR LBD
tetramer (21) and four hRXR DBDs (23). Grid space is 2 nm.
b and c are the same model rotated
counterclockwise by 90° around the z- and
y-axes, respectively.
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Ab Initio Shape Determination Using DAM Technique
(DAMMIN)--
The particle shapes were also computed by the second
ab initio procedure using the DAMMIN program (31). In this
method, a sphere of diameter Dmax is filled with
densely packed small spheres (dummy atoms) with radius
r0
Dmax. The method
searches to minimize differences between experimentally determined
scattering curves and those calculated from DAM models using simulated
annealing algorithm. The "looseness" penalty term ensures that the
procedure yields a compact and interconnected model. The models were
derived from the experimental data assuming a 2-fold symmetry for the dimer and p222 symmetry for the tetramer. The symmetry
restrictions resulted in a significant reduction in the number of
free parameters of the models. The search volume for hRXR
dimer has
been filled with NDAM = 2718 dummy atoms with a
packing radius ra = 0.375 nm within a sphere
with the diameter Dmax = 11.5 nm. The
search volumes for hRXR
tetramer have been filled with
NDAM = 3884 dummy atoms with a packing radius
ra = 0.575 nm within a sphere with the diameter
Dmax = 20.0 nm. Forty independent ab
initio simulations were performed. Of those, 395 ± 10 dummy
atoms were attributed to the final model of hRXR
dimer, and 265 ± 5 atoms were assigned to the final model of the hRXR
tetramer.
The obtained DAMs are presented in Figs. 4 and 5. DAM-derived structure
parameters also agree with the experimental values (Table I). To verify
the uniqueness of the shape restoration using DAM, several independent
restorations were performed using different starting conditions
yielding reproducible results. Maximum concentrations of the protein
used for SAXS solution studies of the hRXR
dimer (3.1 mg/ml) and the
hRXR
tetramer (14 mg/ml) limited the maximum resolution of the final
models to 3.0 and 2.1 nm, respectively (Table I). These resolutions do
not permit unambiguous determination of the spatial positions of their
secondary structure elements, but allowed us to obtain the overall
shape of the molecules and relative position of their individual
domains
hRXR
Dimer and Tetramer Solution Structure--
The best
fitting SAXS hRXR
AB dimer model, constructed as described in the
preceding two sections, is shown in Fig. 4. The dimer is an anisometric
U-like-shaped molecule with two clearly developed substructures that
correspond to homodimers of the LBD and DBD. The two LBDs form the
plate-like base of the U, and each LBD is connected by a long
-helical peptide, protruding like an arm, to its DBDs placed at the
extreme of the U (Fig. 4). The LBDs dimerize through the interface
described in the hRXR
crystal structure (19, 20), involving contacts
between H9 and H10. The two long
-helices connecting LBDs to DBDs
are slightly twisted around the 2-fold symmetry axis of the molecule.
The DBDs form an angle of ~10 degrees in and out of plane of the LBDs
but adopt a position that is relatively close together in a head to
head orientation. The distance between the DBDs in solution is slightly over 2 nm, which is comparable to the distance between half-sites of a
DR-1 element (3 nm). However, since these are placed in a head to head
orientation, rotation of one of the DBDs by about 180° would be
required for the homodimer to bind to a DR-1 element. Assuming that
this rotation could occur, the data are consistent with the notion that
the hRXR
AB homodimer that we observe in solution is capable of
binding to its cognate DNA response element.
The hRXR
tetramer is an oblate and more elongated X-shaped molecule
(Fig. 5 and Table I). Like the dimer, the tetramer contains well
developed substructures that correspond to the LBDs and DBDs (Fig. 5).
However, in the tetramer, the LBDs comprise the center of the molecule
and the four DBDs protrude from the center. The relative position of
the LBDs is consistent with previous crystallographic and solution
studies of the hRXR
LBDs, which show that the tetramer is composed
of a dimer of dimers where the LBDs of one dimer are in head to head
arrangement with the LBDs of the other dimer. To fit better
experimental scattering curves we allowed for relative rotation of
homodimers around their 2-fold symmetry axis and for adjustment of the
distance between their centers of masses. The model is consistent with
the idea that individual dimer pairs utilize the previously defined
interface that overlaps the intersection of H9 and H10 and that the
extensive tetramerization interface utilizes interactions between
H3-H3, H11-H11, and H12 with the H3-H5 hydrophobic cleft region in the
adjacent dimer (21), although limited resolution of SAXS models does
not allowed detailed description (Fig. 5). However, in striking
contrast to the DBD-LBD homodimers, the DBDs of the tetramer were not
close together (Figs. 4 and 5). Individual distances between the DBDs
of each dimer pair were 6 nm. This suggests that the DBDs of the RXR
tetramer would not be able to interact with the DR-1 element without
significant conformational changes. Thus, the x-ray solution studies of
the RXR tetramer present an explanation why it may not bind well to DNA.
 |
DISCUSSION |
Small-angle x-ray scattering analyses yield data that permits
assembly of structural models of protein in solution. The technique has
some obvious disadvantages, especially relative to x-ray
crystallography. First, the data is collected at relatively low
resolution (nm range relative to angstrom range) and therefore does not
permit accurate determinations of the position of individual amino
acids and their side chains within the structure. Second, the technique relies on modeling of relatively low amounts of data and therefore depends upon assumptions about the tertiary and quaternary structure of
the protein and internal symmetries of the overall structure (see
"Materials and Methods"). Should these assumptions prove incorrect,
then the deduced structures may prove to be inaccurate. Nevertheless,
the technique is useful for less detailed descriptions of overall
protein conformations and, further, can be used in conjunction with
published high resolution crystal structures to infer the likely
conformation of proteins in solution. In this sense, small-angle
scattering may have some advantages over x-ray crystallography, which
provides a high resolution "snapshot" of a particular conformation
within a crystal lattice, which might be influenced by the crystal
packing and can not always be readily interpreted in terms of the
oligomeric units of the protein in solution.
In this study, we reported low resolution synchrotron small-angle
scattering structures of unliganded hRXR
AB oligomers. We employed
two independent ab initio shape restoration methods to
obtain a molecular envelope of RXR
in its dimeric and tetrameric forms to 3.0 and 2.1 nm, respectively. The two ab initio
shape restoration methods differ in that SASHA modeling yields an
overall molecular envelope, whereas DAMMIN modeling provides a dummy
atoms model. Figs. 4 and 5 demonstrate that the dummy atoms derived from the DAMMIN model are highly dense inside the envelope derived from
the SASHA model, whereas density of dummy atoms outside it is low.
Given the differences in the number of adjustable parameters (Table I),
the methods therefore agree well within the limits imposed by the
intrinsically low resolution of SAXS. Our models for dimer and tetramer
forms indicate that both oligomers are relatively elongated and further
suggest that the dimer adopts an oblate conformation, whereas the
tetramer adopts an X-shaped conformation. The Stokes radii computed on
the basis of the proposed low resolution models for both oligomers
agrees well with the experimental measurements of Stokes radii
independently obtained by size exclusion chromatography and dynamic
light scattering studies (see "Results"), providing independent
biochemical verification of our models.
In an attempt to understand the divergent nature of the dimer and
tetramer shapes, we positioned published crystallographic models of the
hRXR
DBD fragment (23) and hRXR
LBD homodimers/homotetramers (21)
within the ab initio dimer and tetramer structures. Internal P2 and P222 symmetries were imposed in the case of the dimer and the
tetramer, respectively (Figs. 4 and 5). These symmetries correspond to
the internal symmetry of the previously determined RXR
LBD crystal
structure (21) and were also previously confirmed by solution
scattering studies of the same LBD protein (20). Given these
constraints, the crystallographic models show a relatively good fit
into the dimer and tetramer envelopes. The likeliest hRXR
AB dimer
conformation is a U-shaped structure with the two DBDs occupying the
tips of both arms of the U. The DBDs are closely positioned in such a
way that they could contact DNA without major conformational changes of
the protein. However, as discussed in the Introduction, RXR homodimers
preferentially bind to DR-1 elements containing DNA half-sites spaced
as direct repeats. Thus, if the LBDs are dimerized as shown in this
structure and by mutational analyses one of the DBDs would have to
rotate by ~170-180° for the homodimer to bind to DNA.
Alternatively, the DBD-LBD conformation that is revealed in this
structure could bind DNA if one of the DBDs were to bind
nonspecifically to one of the half-sites. By contrast, the hRXR
AB
tetramer adopts an X-shaped structure in solution. The LBDs are known
to tetramerize by forming a head to head arrangement of paired dimers
(dimer of dimers) with extensive contacts between H3-H3, H11-H11, and
H12 with the coactivator cleft of the neighboring molecule. The
likeliest tetramer structure fits the head to head arrangement of LBD
homodimers into the middle of the X shape, and the DBDs would occupy
the tips of the four points. Interestingly, the DBDs of the tetramer
would be widely spaced in this configuration and therefore unlikely to
bind DR-1 elements with high affinity without major conformational
changes. Thus, we propose that the dimeric and tetrameric forms of the LBD differentially position the RXR DBDs, suggesting that RXR tertiary
structure is sensitive to alterations in its quaternary state. These
low resolution structures represent the first models proposed for a
nuclear receptor containing both a DBD and LBD. Moreover, our models
suggest an explanation for the observed inability of the unliganded
tetramer to bind to DNA.
Although these results cannot absolutely assure that other models would
not give a better fit, we believe that the obtained solutions must be
close to reality. First, the molecular envelopes obtained by two
independent shape restoration methods (SASHA and DAMMIN) give similar
solutions and the deduced Stokes radii from the models correspond well
to the observed Stokes radii. Second, the DBD and LBD crystal
structures fit well into the overall molecular envelopes. Third, the
maximum dimensions retrieved from the p(r) function impose rather
elongated shapes that are hard to explain by alternative placements of
RXR LBDs and DBDs without invoking conformations that contradict
previous results from high resolution crystallography and molecular
genetics. Nevertheless, while the majority of dummy atoms derived from
the DAMMIN modeling technique fit into the molecular envelope derived
from the SASHA modeling technique, we recognize that there are
discrepancies in the placement of some DAMs (Figs. 4a
and 5a). These differences could reflect a natural level of
error in modeling techniques or uncertainties in the structure of the
molecule in solution at these locations. We can not distinguish these
possibilities, but it is interesting that the greatest disagreements in
the dimer structure lie around the upper part of the LBD around the
predicted position of H12 and the greatest disagreements in the
tetramer structure lie close to the predicted position of the DBDs. It
is intriguing to suggest that these discrepancies between techniques
may reflect relative mobility of these regions of the protein in solution.
While solution x-ray studies can tell us that the relative orientation
of the LBD and DBD may differ in the dimer and tetramer forms, the
relatively low resolution of the structures prevents us from learning
why. We suggest that differences in the conformation of the LBDs in the
dimer and tetramer form alter the relative position of the H1/hinge
region and reposition the DBDs. However, these subtle changes, if they
exist, would be too small to be perceived in solution x-ray scattering.
Nonetheless, we note that several distinct lines of evidence indicate
that nuclear receptor LBDs must be able to influence the behavior of
the DBD. For instance, RXR homodimer binding to DR-1 elements is
enhanced by hormone. Thus, ligand-induced conformational changes in the
LBD must improve the fit between hRXR and DR-1 and increase the
affinity of the interaction. Likewise, TR homodimer binding to DR-4 and
to an inverted palindromic element (F2) is reduced by hormone
(43-45). In the case of TR homodimers, we hypothesize that
ligand-induced conformational changes alter the relative position of
the TR DBDs and thus reduce the fit of the TR for both types of
response element. It will be important to obtain crystal structures of
liganded nuclear receptors containing more than one domain and bound to a variety of DNA elements to understand these phenomena.
The dynamic equilibrium between RXR oligomers is influenced by receptor
concentrations, ligand, and DNA (14, 15). Thus, it seems plausible that
sequestration of RXR in tetramers in vivo could provide
a mechanism for the cell to store an excess of receptors until
physiological functions related to RXR homodimer and heterodimer activities are called into action (14, 15). In addition to the
inactivation revealed by the current studies through separation of the
RXR DBDs, a second mechanism for inactivation is provided by structural
studies of the RXR LBD tetramer (21). In this case, H12 of one receptor
binds to the H3-H5 region of the neighboring molecule, an interaction
that is partially analogous to the way that coactivator peptides bind
to the nuclear receptor hydrophobic cleft. Placement of H12 from one
receptor into the H3-H5 region of the other receptor prevents
coactivator binding by occluding the hydrophobic cleft. Our recent
studies revealed that the TR and RXR hydrophobic cleft also binds the
corepressor N-CoR, suggesting that the orientation of H12 in the
tetramer could also occlude the RXR N-CoR interaction surface (46).
While our studies have uncovered possible structures of the unliganded
hRXR
AB tetramer, RXR may also be able to form biologically active
tetramers in a different configuration. The cellular retinol-binding protein II (CRBPII) promoter contains two non-optimal DR-1 sites that
are poorly recognized by the RXR dimer, suggesting that the RXR
tetramer is the active species for controlling expression of CRBPII
(47-49). These data argue that RXR tetramers control gene regulation
in vivo through cooperative DNA binding to the CRBPII
promoter (47) and to a wide variety of four differently oriented
half-sites both in vitro and in vivo (49). While
the interactive surfaces leading to the formation of this type of tetramer are also located in the LBD (49), it is likely that this
tetramer is different from the one that we have described here because
hormone-dependent activation of CRBPII gene expression will
require coactivator recruitment, which as discussed above would be
excluded in the tetramer structure that was used to obtain the model
in the current studies. We also stress that our studies exclude the RXR
N-terminal domain. RXR
or RXR
, but not RXR
, can form tetramers
cooperatively on the CRBP-II promoter and regulate this gene
efficiently (49), and the differences between the isoforms maps to the
RXR
N-terminal domain. This suggests the RXR N-terminal domain may
play an important role in regulating formation of the active DNA and
ligand-dependent tetramer.
In conclusion, the low resolution solution structures for the
hRXR
AB region determined in this work provides the first
three-dimensional model for the member of the nuclear receptor super
family containing more then one functional domain. Our studies suggest
a possible explanation for the previously observed differences in DNA
binding activity between dimers and tetramers. The present ab
initio models of hRXR
dimer and hRXR
tetramer also provide a
basis for further analysis of the hRXR
interaction with ligands
and DNA response elements.