From the Genomics and Structural Biology Laboratory, UPR 9004, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, 1 rue Laurent Fries, 67404 Illkirch Cedex, Cité Universitaire de Strasbourg, France
Received for publication, September 29, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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The major postembryonic developmental events
happening in insect life, including molting and metamorphosis, are
regulated and coordinated temporally by pulses of ecdysone. The
biological activity of this steroid hormone is mediated by two nuclear
receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein
(USP). The crystal structure of the ligand-binding domain from the
lepidopteran Heliothis virescens USP reported here shows
that the loop connecting helices H1 and H3 precludes the canonical
agonist conformation. The key residues that stabilize this unique loop
conformation are strictly conserved within the lepidopteran USP family.
The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X
receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of
this receptor provides the template for designing inhibitors, which
could be utilized as a novel type of environmentally safe insecticides.
The Ultraspiracle protein
(USP),1 an orphan nuclear
receptor, is the insect ortholog of the vertebrate retinoid X receptor (RXR). Like RXR, it belongs to the superfamily of nuclear receptors (NR) (1), which are intracellular receptors regulating target gene
expression upon binding of small, hydrophobic molecules like steroids,
retinoids, thyroid hormones, and vitamin D3. In insects, steroid hormones, the ecdysteroids, control insect development, molting, metamorphosis, and reproduction (2-4). The weak binding of
20-hydroxyecdysone, the biologically active ecdysteroid for most
insects, to the ecdysone nuclear receptor (EcR) is dramatically stimulated by addition of USP, resulting in an affinity in the nanomolar range (5). In fact, the functional ecdysteroid receptor is
formed by the heterodimer EcR/USP, as demonstrated in vitro by DNA binding, ligand binding, and transactivation assays (6) and
in vivo on fly usp mutants (7). Like its
vertebrate homolog, USP can heterodimerize with several vertebrate NRs
(5), and in insects, it forms heterodimeric complexes with EcR and also with at least another NR, DHR38 (8). For vertebrates, the
heterodimerization of RXR with several NRs allows the modulation of
their affinity for genomic response elements (9-11).
Unlike its vertebrate homolog RXR for which a ligand is known, the
9-cis-retinoic acid (9-cis-RA), no hormone ligand
has been identified for USP up to now. Juvenile hormone (JH), an
esterified sesquiterpene, has been put forward as the candidate
hormone ligand of USP. In fact, it has been known for a long time that
JH prevents metamorphosis by modulating the ecdysteroid action at the
outset of the ecdysteroid rise for the molt (3, 12-14). The
hypothesis that JH might act through a NR relies on its chemical
analogy with the vertebrate terpenes, represented by the retinoic acid. The idea that USP could be the receptor of JH or any of its derivatives raises the attractive possibility that JH might directly modulate the
activity of the EcR/USP complex (15). In addition, some evidence
was given that JH can bind to USP and stimulate oligomerization of USP
in vitro and in yeast cells (16). However, this is largely debated and still awaits further substantiation (2). In particular, the
dissociation constant for binding of JH to USP was measured to be about
0.5 µM (16). Compared with the typical affinity of
hormones for their nuclear receptor (in the nanomolar range), this low
affinity questions whether such concentrations might be meaningful at a
physiological level, where other cellular molecules might compete with
JH for USP binding.
NRs are modular proteins possessing a highly conserved DNA-binding
domain and a moderately conserved ligand-binding domain (LBD) (17). The
LBD confers specificity to ligand binding and is responsible for
ligand-dependent transactivation. Crystallographic investigations of the LBD structures of several NRs indicate a conserved fold described as an antiparallel In this paper, we report the crystal structure of the LBD of the orphan
nuclear receptor USP of the lepidopteran H. virescens (hvUSP) at 1.65-Å resolution. The ligand-binding domain of hvUSP adopts an antagonist AF-2 conformation that could be driven by the
presence of an unexpected ligand in its ligand-binding cavity. Important structural discrepancies with its vertebrate homolog RXR are
observed. In particular, the connecting loop between helices H1 and H3
(L1-3) turns out to be a key element of this receptor in its actual
conformation. This structural element sterically precludes the agonist
AF-2 conformation and stabilizes an unusual antagonist conformation.
The nature of the ligand observed in the crystal structure of USP LBD
raises fundamental questions about the biological significance of
similar molecules for insect endocrinology.
Protein Expression and Purification--
The H. virescens USP (hvUSP) LBD (residues Val-205 to Met-466) was cloned
as a N-terminal His6-tagged fusion protein in a pET-15b
expression vector and overproduced in the Escherichia coli
BL21(DE3) strain. Cells were grown in 2× LB medium at 37 °C and
subsequently induced for 2 h with 0.8 mM
isopropyl- Crystallization--
Crystallization experiments were carried
out at 4 °C with the hanging-drop vapor diffusion method. The
protein concentration was 3-9 mg/ml. Crystals of 200 × 200 × 400 µm3 were grown in about 10 days from a solution
containing 10% polyethylene glycol 4000, 50 mM Tris
(pH = 7.5), 100 mM NaCl, 5 mM
dithiothreitol equilibrated against a reservoir containing 20%
polyethylene glycol 4000 and 100 mM Tris (pH = 7.5).
Crystals belong to the tetragonal P4322 space group, with
one monomer per asymmetric unit. The unit cell parameters are
a = 58.21 Å, b = 58.21 Å,
c = 144.69 Å, and Data Collection, Structure Determination, and
Refinement--
Crystals were flash-frozen in liquid nitrogen after a
short dip in a solution containing 10% glycerol as cryoprotectant. The native data set was collected on a single crystal on beamline ID14-EH2
at European Synchrotron Radiation Facility (Grenoble, France). Data were processed (Table I)
using HKL programs (30). The crystal structure was solved by molecular
replacement (31) using a partial structure of hsRXR Ligand Characterization--
Time-of-flight mass spectrometry
was utilized either using the electrospray technique (electrospray
ionization) in native and denaturating conditions or the fast atom
bombardment technique. Results showed that a heterogeneous mass
distribution around 740 ± 50 Da was added to the peak
corresponding to the pure protein (30.2 kDa), and the major peak
corresponds to a mass of 745 ± 3 Da. The fatty acid content of
the phospholipid molecules was obtained by organic solvent extraction.
The analysis shows that more than 95% of the fatty acids are composed
of C18:1 (40%), C16:0 (34%), C16:1 (22%); and trace amounts of
C17:0, C18:0, C18:1, and C18:2 are detected as well. In the case of
phosphatidylglycerol molecules, the major species detected by
Time-of-flight mass spectrometry (745 ± 3 Da) correspond to a
molecule with a tail made of C16:0 and C18:1 esterified fatty acids,
fully consistent with the results of the organic solvent extraction and
with the electron density maps.
Structure-based Sequence Alignment--
The present
crystallographic investigations of hvUSP were restricted to the LBD,
which comprises 264 residues starting at Val-205. The sequence of the
hvUSP LBD is shown in Fig. 1 and is
aligned to the sequences of other USP LDBs of the insect orders Lepidoptera and Diptera and to sequences of RXR of isotypes Architecture of USP LBD--
The overall architecture of USP LBD
exhibits the canonical NR fold (18) with 11
The helices H1 and H3 contribute to the outermost shell of the LBD.
They are less coplanar for USP than for RXR
The conformation adopted by the connecting loop L1-3 is unusual and
essential for the stabilization of the actual structure of USP LBD.
This contrasts with the observation that this loop usually behaves, in
most NRs, as a very flexible region. For hsRXR
For the USP LBD, L1-3 adopts none of these conformations. Its path
(Val-220 to Pro-239) was unambiguously inferred from electron density
maps, as demonstrated in Fig.
3A. Only a few residues at the
beginning of the loop, Asp-222, Pro-223, and Ser-224, were included as
alanines due to the weak electron density of their side chains.
Accordingly, the temperature factors of these residues are higher
(60-64 Å (2)) than those of the other amino acids of L1-3 (36 Å (2)
in average over L1-3). The first residues composing L1-3 form a path
that crosses the region of H3 comprising Gln-256 to Val-262. The next
residues (Glu-226 to Pro-234) delineate an extended loop that runs
along H3. Finally, the last five residues of L1-3 (Asp-235 to Pro-239)
form a loop rather similar to the
The observed conformation of L1-3 is relevant to the physiological
state of the receptor, since no direct crystal contacts that could
induce this conformation are observed in the region of L1-3.
Furthermore, in a region farther away from this loop, the USP LBD and
one of its symmetry-related molecules interact through their
The USP Ligand and the Ligand-binding Pocket--
The unexpected
ligand molecule, which was copurified and cocrystallized with USP LBD,
is a phospholipid. A phosphatidylglycerol or a phosphatidylethanolamine
are consistent with the crystallographic data, and the results of the
mass spectroscopic and chemical analysis. In a similar way, recent
crystallographic investigations of the heterodimer RAR
It is interesting to notice that among the 16 residues of RXR
A superimposition of the cavities of USP and RXR
The binding of the ligand in the USP LBD is mostly responsible for the
conformation of the N-terminal part of helix H3, which is significantly
displaced outwards from the protein core compared with the ligand-bound
RXR The Antagonist Conformation of USP LBD--
The structure of the
USP LBD reveals that the helix 12 adopts a conformation observed in the
case of several NRs, such as RXR
The length of helix H12 is identical to that of antagonist RXR The Connecting Region L1-3 Precludes the Agonist
Conformation--
Unlike in all other NR LBDs known up to now, one
structural element, L1-3, plays a crucial role in USP. From the
superimposition of USP and holo-RXR
The loop L1-3 is also involved in contacts with the N-terminal part of
H11 and L11-12 through the residues Gln-228 to Arg-231 and Asp-235 to
Asn-237, as shown in Fig. 3B. The backbone carbonyl group of
Gln-228 is hydrogen-bonded to the side chain of Ala-442, while that of
Phe-229 binds to the NH group of Ala-442. Furthermore, Arg-231 plays a
central role by establishing strong stabilizing interactions with
L11-12: its backbone amide group binds to the carbonyl group of
Leu-440, while its side chain is hydrogen-bonded to the carbonyl group
of His-439 and establishes a van der Waals contacts with Val-441 and
Ala-442. Other interactions involve the backbone carbonyl group of
Asp-235 with the side chain of His-439 and a water-mediated interaction
with Val-441. The hydroxyl group of Ser-236 also makes a van der Waals
contact with the side chain of Leu-440.
It is important to notice the high sequence conservation among all the
residues involved in the interactions of L1-3 with H3 and with
L11-12. In the case of the interactions of L1-3 with H3, the main
interacting residues of H3, Arg-243 and Asn-254, are strictly conserved
for all lepidopteran USPs. Similarly, their interacting partners in
L1-3 (Glu-226, Phe-227, Gln-228, Phe-229, Leu-230, Val-232, Gly-233,
Ser-236, Val-238, Pro-239) are strictly conserved for all lepidopteran
USPs, except Phe-227 and Phe-229, which are replaced by leucine and
isoleucine residues, respectively, in the case of bmUSP (see Fig. 1).
In the case of the interactions of L1-3 with L11-12, apart from
Phe-229 and Asp-235, both sets of interacting residues in L1-3
(Gln-228 to Arg-231, Asp-235, and Ser-236) and in L11-12 (His-439 to
Ala-442 in L11-12) are strictly conserved among all lepidopteran USPs.
These conservations show a strong evolutionary pressure that suggests
the functional relevance of the observed conformation for the
lepidopteran USPs. The other subgroups of the USP family do not exhibit
the same sequence conservation, suggesting the possibility of
alternative conformations.
The final conformation of the connecting loop L1-3 adopted by the
liganded USP LBD most probably acts as a regulator during the
transconformation process of the LBD occurring when the ligand binds.
For RXR Biological Significance and Concluding Remarks--
The ligand
captured inside the ligand-binding pocket of the USP LBD is most
certainly an endogenous molecule of E. coli used as the
protein expression system. The type of ligand found in the
ligand-binding pocket of USP is consistent with the nature of the
endogenous phospholipids of this bacterial organism, where only three
types of phospholipids coexist (phosphatidylethanolamines, 70%;
phosphatidylglycerols, 18%; cardiolipines or
diphosphatidylglycerols, 12%) (39). The phospholipid ligand in the USP
LBD cannot be displaced by competition with juvenile hormone ester, nor
with methoprene, one of the juvenile hormone mimics, as checked by mass
spectrometric studies (data not shown). Although the bound phospholipid
is not the natural ligand, it stabilizes the observed conformation,
thus favoring the ligand-bound conformation versus the less
stable ligand free one.
The large size of the ligand-binding pocket mainly results from the
peculiar positioning of the N terminus of H3 and of the loop L1-3.
This vast hydrophobic cavity, which accommodates a large-sized
phospholipid ligand, is composed of two parts: one containing the
ligand and another one near a conserved arginine. The shape and the
chemical nature of this empty part of the pocket are similar to the
corresponding parts of the RXR
On the other hand, the present data suggest that the actual USP ligand
could resemble the E. coli endogenous phospholipids. One
class of molecules found in insects, the diacylglycerides, are worth
mentioning. These molecules are the primary circulating lipids in
insects (3). In the insect hemolymph, these molecules are bound to
carrier proteins (called lipophorins), which enhance the solubility of
these molecules. Whether these molecules are relevant, directly or
indirectly, to USP cannot be decided from this study.
The functional ecdysone receptor is formed by the heterodimer EcR/USP.
It is therefore instructive to substitute, in the crystal structure of
the heterodimer RXR/RAR, the RXR LBD by the USP LBD. This is
corroborated by the fact that a strong similarity is observed for the
homo- and heterodimer interfaces of most NRs (26). This substitution
shows that the interface is conserved, as already seen from the
sequence alignment (Fig. 1) and suggests that the loop H5-s1, which is
only partly resolved in our structure, would embrace the partner
receptor and make tight contacts with the region L8-9 of the LBD of
the USP partner connecting helices H8 to H9. This interesting feature
remains to be checked in the structure of the functional ecdysteroid
receptor EcR/USP. Most importantly, it will be of high biological
significance to determine whether USP still adopts the observed
antagonist conformation when heterodimerized with agonist-bound EcR.
This would be the first example of a conformational dissymmetry between
the two heterodimeric partners and would imply the modulation of the
response of EcR to agonistic ligands by an antagonist nuclear receptor.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helical sandwich (18)
composed of 11
-helices (H1, H3-H12) and two short
-strands (s1-s2). Most importantly, the crystal structures of the LBDs of
unliganded (apo) (18) and liganded RXR (19) and of other liganded NRs
(for a review, see Refs. 20 and 21) have allowed us to gain insight
into the molecular mechanisms that underlie the dramatic structural
reorganization that accompanies the ligand binding to the LBD. This
conformational rearrangement mostly affects the N-terminal part of H3,
H11, and H12, which carries the autonomous activation function (AF-2)
(18, 19, 22). Upon binding of an agonist ligand, the corepressors are
released, and the LBD adopts a unique conformation that generates an
interaction surface for the coactivators, which then recruit
multiprotein complexes and lead to the activation of responsive genes
(23). In contrast, antagonist ligands induce a transconformation of the
LBD that does not allow binding of coactivators. Several antagonist
conformations have been observed that can be considered variations
around a common theme (24-27). In these cases, the activation helix
H12 lies in a groove similar to the binding site of the helical NR-box module of nuclear coactivators (the so called antagonist groove) (25,
28, 29). In the case of partial agonist/antagonist ligands, helix H12
also lies in this groove, even though the ligands do not sterically
preclude the agonist position of H12. However, in contrast to the full
antagonist ligands, a weak but clear transcriptional AF-2 activity is
reported in the presence of the corresponding ligand as would be in the
presence of an AF-2 full agonist ligand. Partial agonist/antagonist
AF-2 conformations were reported for ER
/genistein (27) and
RXR
/oleic acid (26) LBD structures.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside at 24 °C.
Purification procedures include an affinity chromatography step on a
cobalt chelating column followed by a gel filtration on a Superdex 200 16/60 column. After tag removal by thrombin digestion, protein was
further purified by gel filtration. A homogeneous monomeric species was
observed in solution. Purity and homogeneity of the protein were
assessed by SDS and native polyacrylamide gel electrophoresis,
denaturant, and native electrospray ionization mass spectrometry and
dynamic light scattering.
=
=
= 90°. The solvent content amounts to 32%, and the estimated B factor
by Wilson plot is 27 Å2.
(19) as a search
model. The weak solution was obtained with a correlation of 24.8% and Rfree = 54.5% after rigid-body refinement. The
phasing power of the model was low and required numerous manual
building cycles using O (32). The wARP method (33) was used as a tool
for checking the correctness of the partially built structures.
Refinement was performed with CNS (34) using a maximum
likelihood target and bulk solvent correction. Cycles of manual model
building and least square minimization followed by simulated annealing
and individual isotropic B factor refinement led to the final model. Solvent molecules were located in a Fo
Fc map contoured at 3
. The final model,
refined to 1.65 Å resolution, comprises 246 residues, 259 water
molecules, and one ligand molecule. A large part of the connection loop
between helix H5 and the entry of the
-sheet (residues 306-315) as
well as the C-terminal extension prolonging H12 (residues 459-466)
could not be modeled due to poor electron density in these regions. The
quality of the final model (Table I) was monitored with Procheck
(35).
Crystallographic analysis
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
,
and
. The sequences of USP LBDs altogether are rather well conserved
with respect to those of RXR LBDs (between 34 and 42% sequence
identity). However, while the conservation is highly pronounced within
the whole RXR family, the USP sequences are highly conserved only
inside the lepidopteran family (83-87% sequence identity), those of
the dipteran family being much less conserved when compared with each
other (between 46 and 54% sequence identity) and to the sequences of
the lepidopteran USP LBDs (between 40 and 49% sequence identity). On
the basis of this sequence alignment, the secondary structure elements,
11 helices and a
-sheet can be predicted for USP LBDs using the
canonical structure of NR LBDs (17). The crystal structure of hvUSP LBD
supports these predictions, and the secondary structure elements are
represented schematically in Fig. 1, together with those of hsRXR
LBD. As readily shown in this figure, the helix H3 of hvUSP LBD is one turn longer compared with its counterpart in the RXR
crystal structure. This figure also indicates that most of the conserved residues between lepidopteran USPs and RXRs are located in the helices,
in particular in those forming the core of the LBD as well as within
the signature region (17). Divergence between USPs and RXRs is observed
mainly for two loops that connect helix H5 to the
-turn (s1) (H5-s1)
and helix H1 to helix H3 (L1-3). The loop H5-s1 is longer for USPs
than for RXRs. Its length also varies inside the USP family, and it
shows rather poor sequence conservation. On the other hand, the length
of L1-3 is rather similar for USPs and RXRs. Its sequence is poorly
conserved between the two families. However, it is highly conserved
inside the family of lepidopteran USPs to which hvUSP belongs. This is
remarkable, because within the whole NR superfamily L1-3 is usually
found to be extremely variable in length and in sequence, consistent with its nature of a rather flexible and loosely structured region. In
contrast, the crystal structure of hvUSP LBD presented here shows that
L1-3 behaves as a rather stiff region due to strong interactions with
several key secondary structure elements of the LBD.
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Fig. 1.
Sequence alignment of USP and RXR
ligand-binding domains. The alignment encompasses USP LBDs
from insects of the Diptera and Lepidoptera orders and RXR from various
organisms. Dipteran USPs include those of Chironomus tentans
(ct), Aedes aegypti (ae), and D. melanogaster (dm). Lepidoptan USPs comprise the
sequences of Manduca sexta (ms), Bombyx
mori (bm), Choristoneura fumifera
(cf), and Heliothis virescens (hv).
The sequences of RXR are shown for the three isotypes, ,
, and
, of Homo sapiens (hs), Mus
musculus (mm), and Xenopus laevis
(xl) RXR LBDs. The sequence numbering above and below the
alignment are for hvUSP and hsRXR
, respectively. Identical residues
for the whole alignment are shown in inverted type with a gray
shaded background. Yellow boxes specify residues
conserved for all dipteran and lepidopteran USPs. Conserved residues
between the lepidopteran USP family and the RXR family are highlighted
in red. The residues conserved only in the lepidopteran
family are highlighted in blue and those conserved only in
the RXR family in green. The secondary structure elements
corresponding to the crystal structures of hvUSP and hsRXR
LBDs are
depicted in blue and green, respectively, below
the sequence alignment. The light gray bar above the
alignment specifies residues that superimpose with a
C
-C
distance cutoff of 2.5 Å in these
two crystal structures. Residues interacting with the ligand in USP are
indicated by blue colored dots and those interacting with
9-cis-RA in hsRXR
by green colored dots. This figure was
prepared using ALSCRIPT (40).
-helices (H1, H3-H12)
and two short
-strands (s1-s2). In the following, the structure of
USP LBD will be compared with two other crystal structures that bear
the major features of NRs and are closely related to it: the
agonist-bound (holo) RXR
(hsRXR
/9-cis-RA) (19) (Fig.
2A) and the RXR
in an
antagonist conformation (msRXR
/oleic acid) (26) (Fig. 2B)
LBDs. The superimposition of the USP LBD to the structure of
holo-RXR
LBD was done by a least square fit (using the LSQ options
of O (32)). Overall, the secondary structure elements of the USP LBD
superimpose rather well with those of holo-RXR
LBD. The root mean
square deviation (r.m.s.d.) is 1.22 Å for 183 matched C
's atoms
out of 246. Seven helices match rather well (r.m.s.d.: 1.13, 0.88, 0.57, 1.18, 0.67, 0.69, 0.75 Å for H4, H5, H7-H11, respectively). The
helices H1, H3, H6 and the
-sheet show larger deviations. The
activation helix H12 adopts a conformation similar to that observed in
RXR
in the antagonist conformation. However, USP harbors at the same time features characteristic of agonist-bound NR LBDs, namely the
length of H11 closer to that of agonist-bound RXR
and the positioning of the phenylalanine residues at its C-terminal part. The
coexistence of features related to both agonist and antagonist-bound NR
LBD structures is a unique and remarkable property of USP, which will
be discussed below in more detail.
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Fig. 2.
Stereo views showing the superimposition of
the crystal structure of USP with those of agonist (A)
and antagonist-bound (B) RXR
LBDs.
-Helices are represented as
cylinders and
-sheets as arrows. The overall
structure of USP LBD is colored in dark yellow. Helices are
labeled accordingly. The structure of RXR
/9-cis-RA (19)
is depicted in blue (A) and that of RXR
/oleic
acid (26) in green (B). Notice that for these two
RXR
structures helix H3 is represented by two unconnected tubes to
better account for the considerable bending of the N and C termini
toward the protein core. This figure as well as Figs. 3 and 4 were
generated with the program SETOR (41).
, the angle between their
helical axes being 12.1° larger than the corresponding value in
RXR
. This is clearly correlated to the path adopted by the loop
L1-3 connecting H1 to H3. It induces a considerable difference in the
positioning of the N- and C-terminal parts of H3 compared with agonist
and antagonist-bound RXR
LBDs, resulting in a more straight helix,
as depicted in Fig. 2. The N-terminal region of H3 (Pro-240 to Cys-250)
is displaced outwards the protein core in a substantial manner. It is
tilted by about 24° with respect to the same region in holo RXR
.
This position is intermediary between the positions of the N-terminal
region seen in the apo RXR
and the holo RXR
LBD structures (data
not shown). The outward bending of the C terminus of H3 (by about
10°) has repercussions on the positioning of the neighboring loops,
L3-4 and L8-9. The loop L3-4, which is part of the signature region
of NRs (17), is displaced laterally by about 1.8 Å and bent in
the direction of L8-9, which itself is pushed outwards by about 1.5 Å.
, the crystal
structures of both apo (18) and holo (19) conformations show
substantial differences in the region connecting helices H1 and H3. In
the holo-RXR
LBD structure, L1-3 consists of an extended loop
passing above the
-sheet. The apo form exhibits an additional helix
in this region, which unfolds in the holo form. In the apo to holo
transition, L1-3 also moves substantially. As suggested from the
comparison of the apo-RXR and the holo-RXR and RAR LBD structures,
L1-3 might act as a molecular spring accompanying the conformational
changes which take place upon ligand binding (19, 22). For ligand-bound
RAR
LBD, the conformation of L1-3 is similar to that of
holo-RXR
, except that it contains a C-terminal region forming a
so-called
-loop (22). Interestingly, for ER LBDs, L1-3 follows a
different path than in the retinoic acid receptors (24, 25, 27). It
passes between helix H3 and the
-sheet, tightly packed to the
protein core.
-loop observed in the RAR
LBD
(22). L1-3 adopts a rather tensed conformation, which allows it to
establish direct contacts with residues in helices H3, H11, and H12 and
stabilize them in their actual position (see Fig. 3B). This
is important, because these helices are the structural elements that
are shown to undergo the largest conformational changes upon ligand
binding (19).
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Fig. 3.
The loop L1-3 connecting helices H1 and
H3. A, stereo view of the 1.65-Å resolution
2Fo Fc electron
density map contoured at 1.0 S.D., showing the quality of the data in
the C-terminal region of the loop L1-3. B, detailed view of
the specific interactions of the connecting loop L1-3 with helices H3
and H11. Protein atoms are colored in light gray for carbon,
blue for nitrogen, and red for oxygen. In
B the protein backbone is colored in yellow,
water molecules are drawn as red spheres, and hydrogen bonds
are depicted as green dotted lines. A few key residues are
labeled accordingly.
-sheets. This contact can most certainly take place, because L1-3
does not occupy this region in solution. If L1-3 was forced to swing
during crystallization and move from a conformation similar to that of
RXR
to its actual conformation, then several secondary structure
elements would need to move dramatically. This drastic reorganization
of the whole LBD is very unlikely to happen, especially because L1-3
is located in a region of the LBD where it makes very specific
interactions with neighboring secondary structure elements involving
many conserved residues (see below).
/RXR
LBD
reveal an E. coli-endogenous oleic acid (C18) or a closely
related compound (stearic (C18) or palmitic (C16) acid) in the RXR
subunit (26). It is important to notice that even though this molecule
is not the true ligand for this vertebrate NR, it induces and
stabilizes an antagonist AF-2 conformation, which is most probably
significant of the structure of the antagonist-bound RXR
LBD. In our
case, the best fit of the electron density was obtained with a
phospholipid with a tail composed of two fatty acids of a length
corresponding to 18 and 16 carbon atoms, respectively. The longer fatty
acid has a rather twisted shape with two major kinks, while the other
fatty acid, bent inside the pocket, has a more regular shape. The tail
of the phospholipid is buried inside the ligand-binding pocket, whereas
its head is located at the very entrance of the cavity, as shown in
Fig. 4A. The residues interacting with the phospholipid ligand are denoted by blue
dots in Fig. 1. van der Waals contacts are observed between the
ligand and residues in helices H3, H5, H7, and H11 in the
-sheet and in the loops L1-3, L6-7, and L11-12. One polar residue, Gln-338 (H6)
is hydrogen-bonded to the ethanolamine moiety in the case of a
phosphatidylethanolamine and to the phosphoryl-glycerol moiety in the
case of a phosphatidylglycerol. Almost all of the residues interacting
with the ligand are conserved among the lepidopteran USPs. Exceptions
are Phe-242 replaced by Tyr for other lepidopteran USPs, Met-323
replaced by Ile for bmUSP, and Ser-431 replaced by Cys in msUSP. The
ligand-binding pocket is lined with a few other polar residues, which
do not make contacts with the phospholipid ligand, but which might be
crucial in the interaction with other types of ligands. These residues,
shown in green in Fig. 4A, are located close to
the C-terminal side of H5 (Gln-256 in H3 and Arg-297 in H5), in the
vicinity of H12 (Asn-287 in H5, Ser-428, and Ser-431 in H10 and His-434
in H11), and at the entry of the pocket (Ser-435 and Gln-338 in
H6).
View larger version (50K):
[in a new window]
Fig. 4.
The ligand-binding pocket and the USP
ligand. A, detailed view showing the phospholipid ligand
and the residues inside the pocket. Residues closer than 4.0 Å from
the ligand are depicted in blue and labeled accordingly.
Polar residues inside the cavity, which do not interact with the
ligand, are shown in green and indicated with green
labels. The phospholipid ligand is colored in gray for
carbon, red for oxygen, and green for phosphor
atom. The protein backbone is colored in dark yellow. H3,
H5, H6, H7, H10, and L1-3 are indicated. B, view showing
the location of the phospholipid molecule in the ligand-binding pocket
of USP relative to that of 9-cis-RA in the RXR cavity. The
phospholipid ligand and the 9-cis-RA are colored in
gray and blue. The conserved arginine belonging
to H5 is also shown in this figure as a ball and stick
representation. The color scheme for the atoms is: gray,
carbon; red, oxygen; green, phosphor;
blue, nitrogen. C, a stereo view of the
superimposition of USP/phospholipid and
RXRa/9-cis-RA in the region of the conserved
arginine belonging to helix H5. The protein backbones of USP and RXR
are represented by orange and blue ribbons,
respectively. The phospholipid ligand is depicted in yellow,
and the 9-cis-RA is colored in light gray for
carbon, blue for nitrogen, and red for oxygen.
Residues belonging to RXR
are shown in blue, while those
of USP are colored in light gray for carbon, blue
for nitrogen, and red for oxygen. Water molecules are drawn
as red spheres, and hydrogen bonds are depicted as
green dotted lines. A few key residues are labeled in
blue for RXR
and in black for USP.
LBD,
which are reported to interact with 9-cis-RA (19) (denoted by green dots in Fig. 1), only 4 of the sequence equivalent
residues of hvUSP interact with the phospholipid (Leu-249, Ile-294,
Ser-431, and His-434). The reason for this behavior lies mostly in the different positioning of the ligands in their respective pocket, as
readily shown in Figs. 4, B and C, and 5. The
9-cis-RA is buried deep inside the pocket, where its
carboxylate group establishes a salt bridge with the conserved arginine
residue belonging to helix H5 of RXR
LBD. In contrast, the
phospholipid does not penetrate as deep inside the cavity. As an
illustration, the tail of the longer fatty acid is located
approximately at the position of atom C9 of 9-cis-RA in
RXR
LBD, while the tail of the other fatty acid goes as deep as
about the
-ionone ring of 9-cis-RA (Fig. 4B).
As a consequence, the arginine residue belonging to H5 in hvUSP LBD,
Arg-297, does not participate to the anchoring of the ligand, as it is
observed for holo RXR
(19) and RAR
(36). Despite the fact that it
does not interact with the ligand, this residue adopts a position close
to that of the homologous arginine residue in holo-RXR
, which
definitely differs from the solvent-exposed position of the apo RXR
conformation (19). Instead of making contacts with the ligand, Arg-297
is hydrogen-bonded to the backbone carbonyl group of Leu-325
(
-sheet) and involved in a water-mediated hydrogen bond network
comprising the carbonyl group of Leu-290 (H5) and the side chain of
Gln-256 (H3) (Fig. 4C). In particular, two of the structural
water molecules, which are involved in these interactions, are
positioned about where the two oxygen atoms of the carboxylate group of
9-cis-RA in RXR
LBD would be located, as can be seen in
Fig. 4C. The side chain of Gln-256 is positioned inside the
ligand-binding pocket with respect to the position of the homologous
residue in RXR
, but it could re-orientate as in RXR
to allow a
carboxylate group or another functional group to interact with
Arg-297.
LBDs is depicted in
Fig. 5. The probe-occupied volume of the
USP ligand-binding pocket, as calculated by VOIDOO (37), is about a
factor 2.5 larger than that of hsRXR
LBD (1256 Å (3) for hvUSP
compared with 489 Å (3) for hsRXR
(19) but comparable with the size of the peroxisome proliferator-activated receptor-
ligand
cavity (~1300 Å (3)) (28). The large size of the ligand-binding pocket of USP is a consequence of its unusual topology. In fact, the
USP cavity is composed of a part that is similar to the ligand-binding pocket of RXR
, although slightly wider, and a bulky tube, which extends to the solvent-exposed region between L1-3, H3, H6, and H11.
For RXR
, the ligand-binding pocket is closed by essentially three
hydrophobic residues (corresponding to Val-246 (H3), Val-341 (L6-7),
and Phe-439 (H11) in hvUSP), while for USP, this region is occupied by
the ligand. The USP cavity is widely open with a large cleft between H3
and H6. For RXR
and other NRs, this region forms tight contacts with
the connecting loop L1-3. Notice that a largely open ligand cavity has
also been observed for the ER complexed to the antagonist hormone
raloxifene (24).
View larger version (57K):
[in a new window]
Fig. 5.
View of the superimposition of the
ligand-binding pockets of USP/phospholipid and
RXR /9-cis-RA.
The ligand cavities of USP and RXR
are depicted in
light blue and magenta, respectively. The
occupation of both cavities by their respective ligand is shown by
transparency. The probe-occupied ligand-binding cavity is calculated by
MSMS with a probe radius of 1.4 Å. The oxygen and phosphorus atoms are
colored in red and light gray, respectively, and
the carbon atoms are colored in yellow for the phospholipid
ligand and in green for the 9-cis-RA. The
position of a few structural elements of USP is indicated by labels.
This figure was prepared using the program DINO (42).
. This shows the adaptation of the N-terminal end of H3
accompanying the binding of the ligand and suggests the mechanism of
the ligand entry in the cavity. For RXR
, the comparison of liganded
versus unliganded conformations indicates a substantial
movement of H3 upon ligand binding in an induced-fit mechanism to clamp
the ligand and lock it inside the pocket (19, 22). In the case of USP,
the displacement of the N terminus of H3, which accompanies the binding
of the bulky phospholipid ligand, results in a more widely open clamp.
As it is apparent from the structure, the phospholipid most likely
penetrates the ligand cavity from the channel formed by helices H3, H6,
H11, and loops L1-3 and L6-7. This is in agreement with the path
followed by the 9-cis-RA when entering the receptor cavity
as described for RXR
(19, 22, 38). According to this dynamic model, the ligand would enter the cavity through a cleft formed by the breathing of H3 and H10/H11 and then be trapped in the observed antagonist conformation.
/oleic acid (Fig. 2B),
RAR
/BMS614 (26), and antagonist-bound ER LBDs (24, 25, 27). The
groove in which H12 lies is the binding site for the helical NR-box
module of nuclear coactivators with a consensus sequence
LXXLL, as shown for the LBDs of peroxisome proliferator-activated receptor-
(28), TR
(29) and ER
(25). Similarly, in hvUSP, Ile-450, Ala-453, and Leu-454 of H12 lie in
approximately the same relative locations as the first, second, and
third leucine residues of the LXXLL binding motif
(IXXAL instead of LXXLL). As for other antagonist
NR conformations, helix 12 packs on a groove formed by residues from
helices H3, H4, and the loop L3-4 (Val-261, Arg-265, Met-275, Glu-276,
Ile-279, Ile-282, Lys-283). However, for USP, the loop L1-3 also
participates in the groove topology, where a few residues (Phe-227,
Gln-228, and Phe-229) make van der Waals contacts with H12.
LBD.
However, the structural principle encountered in other antagonist NR
LBDs, by which helix H11 unwinds to allow H12 to bind to the
coactivator NR box LXXLL motif binding groove, does not fully apply in
the case of the USP LBD. Helix H11 is more in the continuity of H10 and
superimposes very well to helix H11 of the agonist-bound RXR
LBD,
being only two residues shorter than in this case. It follows that the
region connecting H11 to H12 is 6 residues long (His-439 to Thr-444).
These amino acids span a 12-Å strand, resulting in an extended
conformation for L11-12. The C terminus of H11 contains 3 phenylalanine residues, which are conserved in RXR
. In the case of
apo RXR
, the side chains of the first two phenylalanine residues
fill the hydrophobic ligand-binding pocket, and the third one is
solvent exposed, whereas in the agonist form they exchange their roles
(19). The situation in the USP LBD is similar to that of agonist RXR
LBD: Phe-436 and Phe-437 are exposed to the solvent, while Phe-438
contributes to the binding pocket. The side chain of Phe-438 is only
slightly rotated with respect to its counterpart in RXR
to face the
ligand at the level of its shorter fatty acid. For the antagonist-bound RXR
LBD (26), only the first of these three phenylalanine residues belongs to H11. The other two residues which are part of L11-12 contribute to the ligand-binding pocket and in this antagonist conformation would make a steric clash with the phospholipid ligand.
LBDs (Fig. 2A), it
can be observed that a few residues of hvUSP L1-3 (Asn-237, Ser-236,
and Phe-229) lie in approximately the same locations as residues
belonging to L11-12 of holo-RXR
(Asp-444, Thr-445, and Phe-450,
respectively). This comparison leads to a very intriguing consequence:
in its actual conformation, L1-3 precludes the existence of the
agonist conformation, since it would sterically interfere with the loop
L11-12. The steric hindrance for H12 to adopt the agonist position is
therefore due to a constitutive element of the USP LBD structure. The
connecting loop L1-3 stabilizes the N terminus of H3 through a
hydrogen bond network with Arg-243 and Asn-254 of H3, as depicted in
Fig. 3B. The guanidinium group of Arg-243 is anchored by
strong hydrogen bonds to the backbone carbonyls of Gly-233, Ser-236,
and Val-238 and exhibits a van der Waals contact with the side chain of
Val-232. In addition, the backbone amide group of Arg-243 is
hydrogen-bonded to the carbonyl group of Pro-239. For Asn-254,
its side chain makes direct hydrogen bonds with the backbone carbonyl
group of Leu-230, the amide group of Phe-229, and to the side chain of Gln-228. The backbone carbonyl group of Asn-254 is hydrogen-bonded to
the side chain of Glu-226.
, it has been described as a dynamic region acting as a
molecular spring to accompany the considerable movement of helix H3
upon ligand binding (19). For USP, L1-3 adopts a totally different
conformation from that observed in liganded RXR
, and we cannot
exclude this conformation to be different upon binding of a different
ligand. However, the observed conformation of the loop L1-3 of USP is
likely to reflect a peculiar and specific role played by this
structural element inside the family of lepidopteran USPs.
pocket. The guanidinium group of this
residue could interact with a ligand functional group, thereby acting
as a RXR-like ligand anchoring part. It seems likely that the pocket
could hold a different ligand in the region unoccupied by the
phospholipid. This would result in a shrinkage of the cavity to a
volume closer to that of the RXR
cavity. However, in this case, it
is not possible to predict which kind of conformation the LBD would
adopt. Identification of JH as the USP ligand is an elusive issue. In
the case of the juvenile hormone I (3), docking of the acidic form of
the molecule into USP shows that this molecule fits very well inside
the USP cavity, being much less constrained than inside a RXR-like
cavity. Furthermore, the hydroxyl group of Ser-431 located close to the N terminus of H5 could most likely be involved in stabilizing interactions with the ligand. It is interesting to notice that for the
dipteran Drosophila melanogaster and Chironomus
tentans, the arginine residue of H5 is not conserved: it is
replaced by a cysteine and a methionine residue, respectively (Fig. 1).
This suggests a different role for these residues possibly related to a
different type of ligand.
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ACKNOWLEDGEMENT |
---|
We thank Drs. B. P. Klaholz, J.-M. Wurtz, and G. Richards for valuable comments and careful reading of the manuscripts; Dr. J. Fagart for his help with the sequence alignment; Dr. P. Egea for help with the program DINO; and Dr. A. Mitschler for technical assistance and for help with crystal preparation and data collection. We thank the staff of European Synchrotron Radiation Facility beamline ID14-EH2 (Grenoble, France) for assistance during synchrotron data collection. We acknowledge valuable discussions with Drs. A. Podjarny and W. Bourguet. We are grateful to Dr. E.-M. Franken (Bayer AG) for providing us with the hvUSP pET-15b plasmid, Drs. N. Potier and A. Van Dorsselaer for mass spectrometry measurements, and C. Leray (CRTS, Strasbourg, France) for the organic solvent extraction.
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FOOTNOTES |
---|
* This work was supported by BAYER AG and by grants from the CNRS, the INSERM, the Hôpital Universitaire de Strasbourg, and the Ministère de la Recherche et de la Technologie.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.
The atomic coordinates and structure factors (code 1G2N) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org).
To whom correspondence should be addressed. Tel.:
33-3-88-65-33-51; Fax: 33-3-88-65-32-76; E-mail:
moras@igbmc.u-strasbg.fr.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M008926200
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ABBREVIATIONS |
---|
The abbreviations used are: USP, ultraspiracle protein; EcR, ecdysone receptor; RXR, retinoid X receptor; NR, nuclear receptor; AF-2, activation function 2; ER, estrogen receptor; LBD, ligand-binding domain; JH, juvenile hormone; 9-cis-RA, 9-cis-retinoic acid; RAR, retinoic acid receptor; r.m.s.d., root mean square deviation.
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