Modulators of the structural dynamics of the retinoid
X receptor to reveal receptor function
Virginie Nahoum*†, Efre ´n Pe ´rez‡, Pierre Germain§, Fa ´tima Rodrı ´guez-Barrios‡, Fabio Manzo§¶, Sabrina Kammerer§?,
Geraldine Lemaire§, Oliver Hirsch§, Catherine A. Royer*†, Hinrich Gronemeyer§**, Angel R. de Lera‡**,
and William Bourguet*†**
*Institut National de la Sante ´ et de la Recherche Me ´dicale, U554, 34090 Montpellier, France;†Universite ´ Montpellier 1 et 2, Centre National de la Recherche
Scientifique, Unite Mixte de Recherche 5048, Centre de Biochimie Structurale, 34090 Montpellier, France;‡Departamento de Quı ´mica Orga ´nica, Facultad de
Quı ´mica, Universidade de Vigo, 36310 Vigo, Spain;§Institut de Ge ´ne ´tique et de Biologie Mole ´culaire et Cellulaire, BP 10142, 67404 Illkirch Cedex, France;
and¶Dipartimento di Patologia Generale, Seconda Universita ` degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy
Edited by Keith R. Yamamoto, University of California, San Francisco, CA, and approved September 10, 2007 (received for review June 7, 2007)
Retinoid X receptors (RXR?, -?, and -?) occupy a central position in
the nuclear receptor superfamily, because they form heterodimers
with many other family members and hence are involved in the
control of a variety of (patho)physiologic processes. Selective RXR
ligands, referred to as rexinoids, are already used or are being
developed for cancer therapy and have promise for the treatment
of metabolic diseases. However, important side effects remain
associated with existing rexinoids. Here we describe the rational
design and functional characterization of a spectrum of RXR
modulators ranging from partial to pure antagonists and demon-
strate their utility as tools to probe the implication of RXRs in cell
biological phenomena. One of these ligands renders RXR activity
particularly sensitive to coactivator levels and has the potential to
act as a cell-specific RXR modulator. A combination of crystallo-
graphic and fluorescence anisotropy studies reveals the molecular
details accounting for the agonist-to-antagonist transition and
provides direct experimental evidence for a correlation between
the pharmacological activity of a ligand and its impact on the
structural dynamics of the activation helix H12. Using RXR and its
cognate ligands as a model system, our correlative analysis of 3D
structures and dynamic data provides an original view on ligand
actions and enables the establishment of mechanistic concepts,
which will aid in the development of selective nuclear receptor
crystal structure ? ligand design ? nuclear receptor ? agonist ? antagonist
part of multiprotein complexes. These complexes correspond to
chromatin-modifying and transcription-initiating machineries
that act at target gene promoters in a precisely timed and
sequential fashion (1). The binding of a ligand to the ligand-
binding domain (LBD) of NRs constitutes the initial step of this
regulatory process. In this context, the C-terminal helix H12 of
bound ligand, determines the type of coregulator recruited by
the receptor (2). Structural studies have shown that in agonist-
bound NR LBDs, H12 adopts the so-called ‘‘active’’ or ‘‘holo’’
conformation and provides a binding surface for short NR
interaction motifs of coactivators (3). In contrast, antagonists
prevent H12 from adopting the holo position (4).
Therapeutically, retinoid X receptor (RXR)-selective ligands,
referred to as rexinoids, are used in cancer therapy, and previ-
ously uncharacterized rexinoid-based therapeutic paradigms are
currently being explored. In addition, rexinoids have promise for
use in the therapy of metabolic diseases (5, 6), but important side
effects associated with existing compounds limit their use.
Improved understanding of the biological role and the structural
biology of RXR (7, 8) will allow the synthesis of selective
Here, we describe the rational design and functional character-
uclear Receptor (NR)-controlled gene expression relies on
a mechanism in which NRs recruit coregulators that are
ization of a spectrum of RXR modulators and discuss the
opportunity to use these ligands as pharmacological tools.
Moreover, using x-ray crystallography and fluorescence anisot-
ropy, we elucidate the molecular basis of their mechanisms and
suggest a structural and dynamic model of partial agonist action.
Results and Discussion
Rational Design of RXR-Selective Modulators. Contrary to the fairly
large collection of existing RXR agonists (9–11), only a few
antagonists have been reported (12–16). To develop RXR
modulators, we selected as a lead compound CD3254 (com-
pound 1), a potent and selective RXR agonist (17) that contains
two positions suitable for chemical modification located in ortho
position to the biaryl bond (Fig. 1). To guide rational ligand
design, we built a model of RXR? LBD bound to compound 1
and identified the methyl group as the most appropriate position
for substitution [supporting information (SI) Fig. 6]. Replace-
ment by a side chain of six atoms was predicted to prevent H12
from adopting the active position and hence generate an RXR-
selective antagonist. The corresponding synthetic route (SI Fig.
7) provided a series of alkyl ether analogs with chain lengths
ranging from C1 to C6 (Fig. 1).
Conversion of CD3254 into Partial and Full RXR Antagonists. Tran-
sient transactivation experiments (Fig. 2A) revealed a progres-
sive transition from agonist via mixed agonist/antagonist to full
antagonist upon extension of the aliphatic side chain. Indeed, 2a
and 2b are progressively weaker agonists that exhibit some
antagonist activity relative to CD3254 (compound 1), whereas 2c
induces only ?15% of the transactivation seen with the parent
compound but acquires strong antagonist activity. Further ex-
tension of the side chain reduces the transactivation capacity of
Author contributions: V.N., E.P., and P.G. contributed equally to this work; P.G., H.G.,
W.B. performed research; E.P. contributed new reagents/analytic tools; P.G., C.A.R., H.G.,
A.R.d.L., and W.B. analyzed data; and H.G., A.R.d.L., and W.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
TIF2 NR2, transcriptional intermediary factor 2 NR box 2; ATRA, all-trans retinoic acid; APL,
acute promyelocytic leukemia; RAR, retinoic acid receptor.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 2P1T, 2P1U, and 2P1V).
?Presentaddress:Eidgeno ¨ssicheTechnischeHochschuleZu ¨rich,Institutfu ¨rMolekularbiolo-
gie und Biophysik, Eidgeno ¨ssiche Technische Hochschule Hoenggerberg, 8093 Zu ¨rich,
**To whom correspondence may be addressed. E-mail: firstname.lastname@example.org, qolera@
uvigo.es, or email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0705356104 PNAS ?
October 30, 2007 ?
vol. 104 ?
no. 44 ?
the rexinoid such that 2e is transcriptionally inactive. However,
2e and 2f retain RXR interaction capacity and consequently act
as RXR antagonists. Coregulator recruitment assays confirm
this transition from agonist to antagonist (Fig. 2B). Although
agonist 1 induces TIF2 coactivator association, 2c does so only
weakly, and 2e is inactive. The antagonist 2e does not acquire
inverse agonist activity, because there is no gain in SMRT
corepressor binding. The 2e-induced RXR antagonist confor-
mation is insensitive to changes in coactivator expression levels,
whereas the agonist activity of CD3254 (compound 1) and the
weak agonist activity of the partial agonist 2c can be largely
enhanced when the coactivator RAC3 is present at high levels
(Fig. 2C). These results suggest that 2c may act as a cell-specific
RXR agonist or antagonist, depending on the cellular coactiva-
tor expression levels. ‘‘Sandwich’’ two-hybrid experiments show
that, in the context of the retinoic acid receptor (RAR)-RXR
heterodimer, 2e does not affect the corepressor interaction
capacity of the RAR? subunit as, for example, does the RAR
inverse agonist BMS493 or the RAR agonist TTNPB (Fig. 2D).
Impact of Ligand Binding on RXR Structural Dynamics. Because
recent reports suggest that the functional consequences of ligand
binding to NRs are mediated largely through modulation of the
impact of ligands 2a–f on RXR helix H12 mobility. Attaching a
fluorescein moiety to the C terminus of RXR? through intein
chemistry (18, 19) allowed analysis of the dynamic properties of
RXR in RAR?-RXR? heterodimers. Steady-state fluorescence
anisotropy measurements showed that addition of agonist 1
slightly increases anisotropy, indicating stabilization of H12,
presumably in the active conformation (Fig. 3A). In contrast,
binding of ligands 2a–f decreases anisotropy, revealing a higher
mobility of H12 in the presence of these compounds. Moreover,
addition of coactivator transcriptional intermediary factor 2 NR
box 2 (TIF2 NR2) peptide (20) causes a slight dose-dependent
increase of the anisotropy of the heterodimer bound to CD3254
(compound 1), indicating that peptide binding further stabilizes
the active conformation (Fig. 3B). With the mixed agonists/
antagonists 2a–d, addition of peptide strongly increases anisot-
ropy, suggesting that TIF2 NR2 reduces helix H12 mobility by
shifting the equilibrium toward the holo form. Remarkably, at
the highest peptide concentrations, the dynamics of H12 is
comparable to that observed with the agonist-bound protein. In
fail to stabilize H12. Time-resolved fluorescence anisotropy
studies confirm the steady-state observations and reveal that
both the fraction of RXR molecules with a stabilized holo-H12
and the time scale of H12 dynamics depend on the nature of the
bound ligand (SI Table 1). Together, these data provide direct
experimental evidence for the differential effects of various
classes of NR ligands on H12 dynamics. Moreover, they reveal
a mechanism according to which mixed agonists/antagonists can
‘‘sense’’ intracellular coregulator levels and act as cell-selective
modulators with agonist or antagonist properties depending on
the cellular context (21).
alkyl ether analogs 2a–f.
Structures of the agonist CD3254 (compound 1) and of the series of
stably transfected with the reporter recombinant 5xGal4-?Glo-Luc and Gal4-
RXR? were incubated with increasing concentrations of compounds to assess
their RXR agonist potential (Left) or with 10 nM CD3254 (compound 1) and
increasing concentrations of the compounds to assess their RXR antagonist
potential (Right). (B) Mammalian two-hybrid assays were performed in HeLa
cells to assess the influence of both 2c and 2e at 1 ?M on interaction between
RXR and both coregulators TIF2 (coactivator) and SMRT (corepressor) in a
cellular context. (C) Mammalian two-hybrid assays were performed in COS
(D) Mammalian ‘‘sandwich’’ two-hybrid assays in HeLa cells to assess the
influence of ligands on corepressor interaction in the context of the RXR-RAR
heterodimer. Note that BMS493 acts as an RAR inverse agonist by stabilizing
corepressor interaction, whereas the RAR agonist TTNPB destabilizes the
RXR agonist/antagonist potential of new compounds. (A) HeLa cells
www.pnas.org?cgi?doi?10.1073?pnas.0705356104 Nahoum et al.
Structural Basis for Agonist to Antagonist Transition. To gain struc-
ture-based insight into the mechanism of action of these mod-
ulators, we solved the structures of RXR? LBD in complex with
2a, 2b, 2c, and TIF2 NR2 (SI Table 2). The structures display the
canonical agonist conformation with H12 capping the ligand-
binding pocket (LBP) and TIF2 NR2 bound to the so-called
AF-2 surface (Fig. 4A). A particular feature of compounds 2a–f
is the presence of two oxygen atoms attached to the tetrahy-
dronaphthyl and cinnamic acid aromatic rings (Fig. 1). In the
structures with 2a and 2b, these oxygen atoms are involved in a
network of hydrogen bonds that stabilizes a water molecule in a
predominantly hydrophobic environment (Fig. 4C). In both
structures, the water molecule occupies a well defined position,
as indicated by the clear electron density (Fig. 4B) and the
B-factor values of 11.77 Å2and 24.15 Å2(mean B values for all
atoms 15.44 Å2and 25.18 Å2in the 2a and 2b complexes). In
contrast, no electron density is observed for the corresponding
water molecule in the 2c complex. The reason for this absence
is that, to maintain favorable interactions with LBP residues, the
longer side chain of 2c rotates around the oxygen atom by an
angle of 42° and adopts a conformation in which the oxygen
electron lone pairs are unfavorably positioned to be engaged in
a hydrogen bond (Fig. 4D). Comparison of these LBP structures
with that of the previously reported (22) RXR? LBD bound to
the agonist SR11237 reveals some residue reorientations, the
most significant one affecting L436 in H11 (Fig. 4E). To
accommodate the water molecule present in the 2a and 2b
complexes and/or the side chains of 2b and 2c, L436 rotates
toward H12. However, in this conformation, the interaction
distance between the C? atoms of L436 and L455 in H12 (3.38
Å, 3.46 Å, and 3.68 Å in the 2c, 2b, and 2a complexes) is
significantly shorter than the sum of the van der Waals radii for
two interacting methyl groups (3.84 Å) (23), thereby generating
repulsive forces accounting for the destabilization of the holo
conformation in solution. Interestingly, the differences observed
for these interaction distances in the various complexes suggest
that the steric constraints exerted by 2c on H12 are stronger than
those imposed by 2b and 2a. Indeed, functional analyses show
efficient than 2a (Fig. 2A). Together, these data reveal that the
mixed activity of 2a relies on a water-mediated mechanism
involving the repositioning L436 (H11), which, through a steric
clash with L455 (H12), lowers the association strength between
holo-H12 and the LBD surface. With 2b, 2c, and most likely 2d,
a direct interaction with L436 accounts for the destabilization of
H12, the effect being proportional to the chain length. Thus,
L436 and more generally all residues that are in contact with
holo-H12 and whose conformation can be affected by the bound
ligand (i.e., L436 and W305 in RXR?) should be considered as
target residues for the design of new NR modulators. With 2e–f,
for which no crystals could be obtained, modeling studies
indicate that their inhibitory effect results from an interference
of their long side chains with L451 of H12 (SI Fig. 8).
UVI3003 as a Tool to Reveal RXR Function. The availability of a
high-affinity RXR-selective full antagonist provided the possi-
bility to assess the contribution of RXR transactivation in the
context of RXR heterodimers by pharmacological means. Com-
paring the responsiveness of promyelocytic NB4 cells expressing
the PML-RAR? oncofusion protein (24, 25) with that of the
HL60 subclone PLB985 (26) revealed major differences in the
growth and differentiative and apoptogenic response toward
RAR and RXR-selective ligands. Indeed, NB4 cells cease
proliferation (Fig. 5A), differentiate (not shown) and undergo
apoptosis (Fig. 5B) in the presence of an RAR? agonist
(BMS753) alone, whereas PLB985 cells require in addition the
presence of an RXR agonist (SR11237) (Fig. 5 A and B). The
RXR antagonist 2e (UVI3003) fully confirmed these results,
because its addition derepressed PLB985 growth inhibition by
the combined action of RAR and RXR agonists but did not
significantly affect growth inhibition or apoptosis in NB4 cells
exposed to RAR agonists (not shown) or RAR and RXR
agonists (Fig. 5 A and B). No similar critical effect of the RXR
antagonist was seen when F9 embryo carcinoma cells were
differentiated to primitive or parietal endodermal cells using
all-trans retinoic acid (ATRA) or ATRA plus cAMP, respec-
tively, or when 3T3L1 preadipocyte cells were differentiated by
troglitazone (Fig. 5 C and D). Together, these data reveal
UVI3003 (2e) as a tool to test the contribution of RXR to
transactivation by a given RXR heterodimer. This is particularly
important in cases where endogenous rexinoids (27) may con-
tribute to a cell biological or physiological phenomenon, or when
RXR is actively engaged in signaling (28, 29). The comparison
of RXR responsiveness of PLB985 and NB4 cells suggests the
challenging possibility that the PML-RAR? fusion protein may
have obliterated the RXR requirement for apoptosis, even
though the leukemogenic species corresponds to higher-order
heterooligomers composed of acute promyelocytic leukemia
(APL) oncofusion proteins and RXR (30, 31). Indeed, that
RAR? ligands alone are sufficient to induce NB4 apoptosis may
explain the efficiency of the retinoic acid therapy in APL
patients. Moreover, the results obtained with PLB985 cells
suggest that certain non-APL leukemias may benefit from the
combined treatment with RAR and RXR agonists.
Concluding Remarks. Despite the fact that RXR plays a major role
as a promiscuous heterodimerization partner (7, 8), and in
anisotropy. (A) Using an RAR?-RXR? LBD heterodimer in which a fluorescent
values in absence of added ligand (No) and in the presence of saturating
concentrations of the RXR agonist CD3254 (compound 1), mixed agonists/
antagonists 2a–d or antagonists 2e–f. Of note, it is very likely that the
heterodimer used as a reference (No) is not truly unliganded, because it has
been previously reported that bacterially expressed RXR is able to bind en-
of increasing concentrations of the NR interaction motif 2 peptide of the
coactivator TIF2 (TIF2 NR2).
RXR? structural dynamics monitored by steady-state fluorescence
Nahoum et al.
October 30, 2007 ?
vol. 104 ?
no. 44 ?
contrast to the related RAR for which multiple small-molecule
RXR. We have generated selective RXR modulators with
distinct pharmacological profiles. Such ligands that are able to
positively or negatively and in a cell-specific manner modulate
RXR transcriptional activity and thus affect several signaling
RXR signaling networks and its functional implications with
multiple partners. This is of particular interest in the context of
aberrant signaling, as is the case for oncofusion protein–RXR
complexes that cause APL (30). Importantly, several RXR-
based therapeutic paradigms for leukemia have been discovered
(refs. 28 and 29, and H.G., unpublished results), thus empha-
sizing the importance of tools to assess the impact of RXR in
Using fluorescence spectroscopy, we have been able to quan-
tify the impact of ligand binding on H12 motion. The combina-
tion of these dynamic data with the static snapshots obtained
using x-ray crystallography has revealed the mechanism under-
lying the effect of ligands whose activity could not be explained
simply by structural considerations. Indeed, although the struc-
tures of the complexes with 2a, 2b, and 2c display helix H12
locked in the same holo position, fluorescence anisotropy data
show they differ from each other by the ‘‘lifetime’’ of their active
conformation in solution. Interestingly, crystal structures reveal
that these ligands impair H12 mobility indirectly, by modifying
the conformation of a residue (L436), which is involved in a
stabilizing interaction with holo-H12. Because 2a, 2b, and 2c
induce slightly different L436 conformations, their impact on
holo-H12 stability is different, thereby providing a rational basis
for the distinct functional outcome of these mixed agonists/
antagonists. We have presented a comprehensive model for the
molecular mechanism of partial agonists that combines struc-
tural and dynamic concepts. These mechanistic insights gained
using RXR as a model of NR/ligand interaction likely corre-
spond to conserved mechanisms that, we believe, will aid in the
design of selective NR modulators with varying degrees of
agonist or antagonist activities and the promise to separate
therapeutically desired from unwanted toxic effects.
Materials and Methods
Chemistry. Experimental procedures for the synthesis of all new
compounds are available in SI Text.
Transfections and Determination of RXR Activity. Transient trans-
fections and two-hybrid assays were performed as described (32,
33). HeLa cells were established according to Chen et al. (34)
and were used as described in SI Text.
Preparation of the RAR?-RXR? LBD Heterodimer for Fluorescence
Anisotropy Experiments. Human RXR? LBD (residues 223–462)
was expressed in Escherichia coli BL21(DE3) as a fusion with an
inducible self-splicing intein (Sce VMA) and a chitin-binding
domain using the vector pTYB1 (New England Biolabs, Ipswich,
MA). Labeling was performed as described (18, 19). The histi-
dine-tagged LBD of human RAR? (residues 176–421 in a
pET15b vector) was expressed and purified as described for
RXR? LBD in SI Text. Fractions containing RAR? LBD were
pooled, dialyzed against buffer B (10 mM Tris?HCl, pH 8.0/150
mM NaCl/5 mM DTT/1 mM EDTA/10% glycerol), and incu-
bated overnight with the fluorescein-labeled RXR? LBD. The
RAR?-RXR? heterodimer was further purified by using a
superdex 75 26/60 gel filtration column (Amersham Biosciences,
Piscataway, NJ). Fractions containing the purified heterodimer
were pooled and concentrated to 117 ?M. The labeled-
heterodimer concentration was estimated to 1.12 ?M by using
Fluorescence Anisotropy Measurements. Steady-state fluorescence
anisotropy assays were performed with a BEACON 2000 po-
larization instrument (Panvera, Madison, WI) regulated at 4°C.
The labeled-heterodimer concentration was set to 0.725 nM by
red (oxygen atoms) and yellow (carbon atoms) van der Waals spheres. Helices and ?-strands are numbered from N to C terminus. Together, helices H3, H4, and
H12 define the activation function 2 (AF-2) surface to which the TIF2 NR2 peptide is bound. (B) 2Fo?Fcdensity (1?) for the LBP of RXR? bound to 2b. W indicates
network is observed in the complex with 2a (not shown). (D) Superposition of the RXR LBP with 2b and 2c. (E) Comparison with the structure of RXR? LBD bound
to SR11237 (Protein Data Bank ID code 1MVC). To accommodate the particular features of 2a–c, L436 must adopt a conformation that differs from that found
in the presence of the agonist. The dashed line between L436 and L455 indicates a short distance.
Structures of RXR? LBD in complex with partial agonists. (A) Overall structure of RXR? LBD in complex with 2a, 2b, or 2c. The ligand is represented by
www.pnas.org?cgi?doi?10.1073?pnas.0705356104 Nahoum et al.
dilution with buffer B. The excitation wavelength was 490 nm,
with emission measured at 530 nm. The TIF2 NR2 coactivator
peptide (686-KHKILHRLLQDSS-698) was added to protein
samples containing 20 ?M of ligand to a final concentration of
10 ?M, and then the sample was diluted successively with buffer
B supplemented with 0.725 nM of heterodimer and 20 ?M of
ligand. At least three independent measurements were made for
each sample. Time-resolved fluorescence anisotropy data were
obtained as described in SI Text.
Protein Expression, Purification, and Crystallization. Human RXR?
LBD was expressed and purified as described in SI Text. Before
crystallization, the protein was mixed with a 3-fold molar excess
of ligand 2a, 2b, or 2c and a 5-fold molar excess of TIF2 NR2
peptide (686-KHKILHRLLQDSS-698). The complexes were
incubated overnight at 4°C and concentrated to ?15 mg?ml?1.
Crystals were obtained by vapor diffusion at 20°C. The well
buffers contained 16% PEG 3350; 0.1 M Tris?HCl, pH 8.5; 1 M
ammonium acetate (2a); 14% PEG 10000; 0.1 M Tris?HCl, pH
8.5; 1 M ammonium chloride (2b); or 24% PEG 4000; 0.1 M
Tris?HCl, pH 7.5 (2c). Crystals were of space group P43212.
Data Collection and Structure Solution. The protein crystals were
mounted from mother liquor onto a cryoloop (Hampton Re-
search, Aliso Viejo, CA), soaked in the reservoir solution
containing an additional 20% glycerol, and quickly frozen in
liquid nitrogen. Diffraction data were collected by using an
ADSC (Poway, CA) Quantum 4 detector at the ID14-EH2
beamline of European Synchrotron Radiation Facility (France)
for the complex with 2a (1.8-Å resolution) and at the ID14-EH4
beamline of ESRF for complexes with 2b (2.2-Å resolution) and
2c (2.2-Å resolution). Diffraction data were processed by using
suite (36). The structures were solved by using the previously
reported structure 1MVC (22) of which the ligand and the
coactivator peptide were omitted. Initial Fo?Fcdifference maps
had strong signals for the ligands and the TIF2 NR2 peptide,
which could be fitted accurately into electron densities. The
of apoptotic Annexin V-positive/propidium iodide-negative PLB 985 or NB4 cells after treatments. BMS753, SR11237, and 2e was used at 1, 1, and 10 ?M,
respectively. (C) Induction of differentiation of F9 cells into primitive endoderm (Upper) after exposure for 48 h to the RAR agonists TTNPB (10 nM) or ATRA (1
?M) alone or in combination with 2e (10 ?M) or into parietal endoderm (Lower) after exposure for 96 h to 8CPT-cAMP (50 ?M) and TTNPB (10 nM) or ATRA (1
?M), and 2e (10 ?M). (D) Induction of differentiation of 3T3L1 cells after exposure to the PPAR agonist troglitazone alone or in combination with 2e (10 ?M).
UVI3003 (2e) as tool to reveal the role of RXR in cell physiology. (A) Proliferation of PLB 985 or NB4 cells after exposure to the RAR?-selective agonist
Nahoum et al.
October 30, 2007 ?
vol. 104 ?
no. 44 ?
structures were modeled with O (37) and refined with REF-
MAC5 (36) by using rigid-body, least-squares, and individual
B-factor refinements. The final models exhibit very good geom-
etry with 94.9% (2a), 95.4% (2b), and 94.9% (2c) of the residues
in the most-favored regions of the Ramachandran plot and no
residue in the disallowed regions. Experimental electron density
Fo?Fcmaps of all ligands, calculated by using the refined model
with the ligands omitted, are shown in SI Fig. 9.
Cell Culture and Analysis of Apoptosis. F9 cells, grown in tissue
culture plates coated with 0.1% gelatin, and preadipogenic
3T3L1 cells were maintained in DMEM supplemented with 10%
FCS and 1 mg?liter?1gentamicin and 2 mM glutamine. For F9
cells, collagen IV expression was monitored by semiquantitative
RT-PCR (SI Fig. 10 and SI Text). PLB 985 and NB4 leukemia
cell lines were cultured in RPMI medium 1640 supplemented
with 10% FCS/2 mM glutamine/25 mM Hepes buffer/40 ?g?ml?1
gentamycin in a humidified incubator at 37°C and 5% CO2.
Apoptosis of leukemia cells was quantified by propidium iodide-
annexin V double staining on a FACScan (Becton Dickinson,
Franklin Lakes, NJ) flow cytometer.
We thank Jean-Franc ¸ois Guichou, Yvan Boublik (Centre de Biochimie
Structurale, Montpellier, France), and Emmanuel Margeat (Centre de
Biochimie Structurale, Montpellier, France) for help with fluorescence
experiments. We acknowledge the technical assistance of the beamline
managers at the European Synchrotron Radiation Facility (Grenoble,
France). We thank Michelle Lieb and Catherine Huck (Institut de
Ge ´ne ´tique et de Biologie Mole ´culaire et Cellulaire, Illkirch, France) for
their contribution to the NB4 and PLB985 experiments and Claudine
Gaudon and Audrey Bindler (Institut de Ge ´ne ´tique et de Biologie
Mole ´culaire et Cellulaire, Illkirch, France) for the RXR reporter cells
and assistance in the analysis. We thank Galderma (Sophia-Antipolis,
France) for providing the CD3254. This work was supported by funds
from the European Community [QLK-2002-02029 ‘‘Anticancer Retin-
oids’’ and LSHC-CT-2005-518417 ‘‘EPITRON’’ (to A.R.d.L. and H.G.),
the Spanish Ministerio de Educacio ´n y Ciencia (SAF04-07131, FEDER,
to A.R.d.L.); and Juan de la Cierva Contract (to F.R.-B.)], the Institut
National du Cancer (H.G.), and the Ligue contre le Cancer (‘‘Equipe
labellise ´e la Ligue’’) (H.G.).
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