From the GlaxoSmithKline, Discovery Research, Research Triangle Park, NC 27709
Received for publication, March 4, 2003 , and in revised form, May 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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Nuclear receptors regulate transcription through the recruitment of
coactivator proteins to the ligand binding domain (LBD)
(11). Structural and
biochemical studies reveal that the coactivator contains a short
-helical LXXLL sequence (where X = any amino acid),
known as an NR box, that binds the nuclear receptor LBD. The NR box is capped
by a charge clamp on the surface of the LBD formed by a lysine on helix 3 and
a glutamic acid on the C-terminal AF2 helix
(12). Despite the availability
of multiple co-crystal structures of ligand/receptor complexes, the mechanism
by which small molecule ligands activate nuclear receptors is still poorly
understood (13). We have shown
previously that a residue in the AF2 helix of LXR
, Trp-443, plays a
role in the activation of the receptor by oxysterols
(14). Based on these results,
we proposed a model where the AF2 helix was stabilized in its active
conformation by a hydrogen bond from the tryptophan indole NH to the sterol
agonist (14). Interestingly,
both the neutral sterol eCH and the acidic nonsterol T1317 are efficacious
activators of LXR
and LXR
in biochemical and cell-based assays
(Fig. 1, b and
c). To probe the molecular basis by which both sterol and
nonsterol agonists regulate LXR activity, we initiated crystallographic
investigations of both LXR
and LXR
LBD. Diffracting crystals of
the LXR
LBD complexed to eCH and T1317 were readily obtained, allowing
us to determine those structures.
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EXPERIMENTAL PROCEDURES |
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The soluble protein was loaded onto the pre-equilibrated nickelnitrilotriacetic acid affinity column (Qiagen) followed by elution with a 10-column volume 50500 mM imidazole gradient. Peak fractions were pooled and dialyzed versus 10 mM Tris, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 5% glycerol.
Protein was further purified using anion exchange. His-tagged LXR was
digested overnight with thrombin at a mass ratio of 1:500. The digested
protein was diluted to 25 mM salt with Q0 (10 mM Tris,
pH 8.0, 0.1 mM EDTA, 5% glycerol, 5 mM dithiothreitol),
loaded onto the preequilibrated anion exchange column, and eluted by a
20-column 0250 mM NaCl gradient. LXR eluted as two peaks at
150 mM NaCl, which were kept separate for crystallization
trials. The eluted protein was dialyzed versus 10 mM Tris,
pH 8.0, 0.1 mM EDTA, 5% glycerol, 5 mM dithiothreitol,
150 mM NaCl, eCH, and SRC1, or T1317 was added, and the protein was
concentrated to 1214 mg/ml for crystallization trials.
Crystallization and Structure DeterminationCrystallization
trials were carried out using the hanging drop method by mixing 2 µl of
protein solution with 2 µl of well buffer. LXR/T1317 complexes
crystallized from 10 to 20% polyethylene glycol 3350 with 100 mM
concentration of a number of salts, including NaF, KF, NaCl, KCl, sodium
formate, sodium acetate, and potassium acetate at 4 °C. LXR
/eCH/SRC1
complexes crystallized from 10 to 12% polyethylene glycol 33508000 with
0.2 M NaCl at 4 °C. Crystals took 46 weeks to grow.
Crystals were frozen in LN2 after transferring stepwise to a cryo buffer
containing the well buffer with 30% polyethylene glycol 400.
All data were obtained at the Industrial Macromolecular Crystallography
Association Collaborative Access Team (IMCA CAT) 17 ID beam line at the
Advanced Photon Source at a wavelength of 1 Å on a MAR CCD detector.
Crystals of the LXR/T1317 complex diffracted to 2.3 Å and were in
the space group P212121 with unit cell
parameters a = 60.25, b = 82.454, c = 123.175.
Crystals of the LXR
/eCH/SRC1 complex diffracted to 2.7 Å and were
in the space group C2221 with lattice constants of a =
71.17 Å, b = 120.01 Å, c = 147.56 Å. All
diffraction data were integrated and scaled using HKL2000
(15). The LXR
/T1317
structure was solved by molecular replacement using the program AMoRe with a
truncated monomeric form of human retinoic acid receptor-
as a search
model. The LXR
/eCH/SRC1 structure was solved by molecular replacement
using a refined LXR subunit from the LXR
/T1317 structure as a search
model. For both complexes, there were two molecules in the asymmetric unit
related by a noncrystallographic dyad. Noncrystallographic averaging was
utilized during refinement. Structures were subjected to multiple rounds of
building using Quanta and refined using CNX and Refmac5.
Biological AssaysLXR cell-free ligand sensing assays and LXR cell-based transactivation assays were performed as described previously (14).
Chemical CompoundsT1317 and eCH were synthesized as described previously (10, 16). The T-CH hybrid was synthesized from chlol-5-en-24-al by addition of (trifluoromethyl)trimethylsilane catalyzed by tetrabutylammonium fluoride, oxidation with Dess-Martin periodinane, and addition of a second equivalent of (trifluoromethyl)-trimethylsilane catalyzed by tetrabutylammonium fluoride. Analytical data are as follows: 1H NMR (CDCl3, 300 MHz) 5.405.34 (m, 1H), 3.613.48 (m, 1H), 2.360.83 (m, 40H); 19F NMR (CDCl3, 282 MHz) 76.7 (q, J = 9.1), 77.1 (q, J = 9.1); time-of-flight mass spectrometry (EI+) m/e 497 (MH+).
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RESULTS |
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In the eCH/LXR complex, the sterol bound with the A-ring oriented
toward helix 1 and with the D-ring and epoxide tail oriented toward the
C-terminal end of helix 10, Trp-457, and His-435
(Fig. 3a). This
orientation was similar to that predicted in the model of Spencer et
al. (14) and was similar
to that of estradiol, progesterone, and dexamethasone in their complexes with
the estrogen, progesterone, and glucocorticoid receptors. However, the steroid
core of eCH was flipped 180° around its long axis so that the angular
methyl groups pointed in the direction opposite that in the steroid hormones
(Fig. 3a). Although
the epoxide oxygen atom lay adjacent to Trp-457, it actually made its hydrogen
bond with the imidazole ring of His-435. The A-ring hydroxyl formed a hydrogen
bond with Glu-281 on helix 3 and was positioned close to Arg-319 on helix 5.
This is reminiscent of the estradiol/estrogen receptor complex, where the
phenolic A-ring hydroxyl makes strong hydrogen bonds with Glu-353 on helix 3
and Arg-394 on helix 5 (17).
However, in LXR
, the A-ring hydroxyl group lay out of the plane of the
Arg-319 guanidinium group, and despite its favorable distance, cannot make a
hydrogen bond with good geometry. In addition to these polar interactions, eCH
also made extensive lipophilic interactions with the ligand binding pocket,
and its conformation was essentially the same in both subunits. In the
T1317/LXR
structure, the acidic carbinol group lay in approximately the
same position as the epoxide of eCH and also formed a hydrogen bond with the
histidine ring of His-435. However, the nonsterol ligand was observed in
different conformations about the tertiary sulfonamide in the two LXR
subunits. In one subunit, the T1317 adopted a gauche conformation
(Fig. 3a), whereas in
the other subunit, it adopted an anti conformation
(Fig. 3b). As a
result, the nonsterol ligand fit into a position corresponding to the C- and
D-rings of eCH and made weak hydrogen bonds with Ser-278 in each case but did
not reach into the volume occupied by the A-ring of eCH and failed to make
interactions with either Glu-281 or Arg-319
(Fig. 3, a and
b). Despite the differences in the regions of the ligand
binding pocket occupied by the eCH and T1317, all of the amino acids
contacting both ligands are conserved between LXR
and LXR
.
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Mechanism of Ligand ActivationA tryptophan in the
LXR AF2 helix (residue 443) has been shown to be essential for
oxysterol activation of the receptor
(14). In LXR
, the
corresponding residue is Trp-457. In contrast to the published homology model
(14), neither the sterol nor
nonsterol ligand formed a direct interaction with the AF2 helix in the crystal
structure (Figs. 3 and
4). The Trp-457 indole was
oriented such that the nitrogen atom was pointed away from eCH, making it
impossible to form a direct hydrogen bond with the epoxide oxygen. Instead,
both ligands interacted with His-435, which made an edge to face interaction
with the tryptophan on the inner surface of the AF2 helix
(Fig. 4, a and
b). The His-435 side chain had some freedom to rotate
about its C
-C
bond, allowing it to swing the edge of its
imidazole ring across the face of the indole ring. This rotational freedom
lets His-435 interact with either the weakly negative
-electron cloud of
the five-membered pyrrole ring or the more strongly negative
-electron
cloud of the benzene ring
(18). This weak electrostatic
interaction can become a strongly favorable "cation-
"
interaction when the imidazole is positively charged and directed into the
most negatively charged regions of the indole
-electron cloud
(19,
20).
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The hydrogen bonding oxygen atom of the agonist ligand was in approximately
the same location in the eCH and T1317 structures, but the position of the
His-435 imidazole was different depending on the strength of its interaction
with the ligand. In the eCH complex, the N2 atom of His-435 was
positioned to donate a hydrogen bond to the epoxide of the sterol side chain
at a distance of 3.453.50 Å
(Fig. 4c). In this
orientation, the imidazole directed its electropositive C
1 and N
2
hydrogens toward the C
atom and six-membered ring of the tryptophan
side chain, corresponding to the electronegative
-cloud of the indole
(18), providing a mechanism
for the sterol to hold the AF2 helix in its active conformation
(Fig. 4c). The
observation that other ligands with only hydrogen bond acceptors in their side
chains, such as 24-ketocholesterol and dimethyl cholenamide
(7,
14), are also efficacious
activators of LXR suggests that this electrostatic interaction is a viable
mechanism for ligand activation of the receptor.
In the T1317 structure, the imidazole swung 1.3 Å toward the acidic
bis-trifluoromethyl carbinol, bringing the His-435 N2 atom to a distance
of only 2.592.75 Å from the carbinol oxygen
(Fig. 4d). In this
orientation, the imidazole directed its electropositive C
1 hydrogen into
the
-cloud of the indole benzene ring, again holding Trp-457 in the active
position. The imidazole rotation also opened space for a water molecule,
observed in both subunits, that made a hydrogen bond to the backside
N
1, further stabilizing the complex. Hydrogen bond lengths tend to
correlate with their energy
(21), suggesting that the
acidic carbinol group makes a stronger hydrogen bonding interaction than the
epoxide. This is consistent with quantum mechanics calculations, which
indicate that the epoxide oxygen has a relatively weak electrostatic charge as
compared with that on the bis-trifluoromethyl carbinol or even as compared
with an ordinary hydroxyl group (data not shown). The T1317 hydrogen bond may
be further strengthened by partial proton transfer, as can occur when the
pKa values of the partners are suitably matched
(21). In this case, the
bis-trifluoromethyl carbinol group has a pKa of
8.4 in water, relatively close to that of histidine, with a
pKa of 6.57.0. Thus, the His-Trp interface
induced by T1317 may be an example of a cation-
electrostatic interaction
(19,
20). Similar cation-
interactions have been observed to regulate the activity of ion channels
(22,
23) and enzymes
(19,
20). Although the degree of
AF2 stabilization by the His-Trp interactions is believed to be different for
the sterol and nonsterol ligands, the position of the C-terminal helix does
not deviate significantly in the crystallized complexes.
Mutagenesis of the His-Trp Activation SwitchTo explore the
functional differences in the electrostatics of the His-Trp activation switch,
we utilized mutants of LXR that might allow activation by T1317 but not eCH.
Although tryptophan is the optimal pairing for histidine in a cation-
interaction, phenylalanine or tyrosine can also substitute
(18). LXR-GAL4 chimeras were
generated where Trp-443 in LXR
and Trp-457 in LXR
were mutated to
phenylalanine or tyrosine. The transactivation capability of the LXR-GAL4
chimeras was measured by their ability to transcribe a secreted placental
alkaline phosphatase reporter on a UAS-tk promoter in transient
transfection experiments (Fig.
5). As demonstrated previously
(14), neither tryptophan point
mutant could be activated by eCH. However, T1317 activated both the W443F
LXR
and the W457F LXR
mutants. We interpret these data as
evidence that phenylalanine can substitute for tryptophan in the electrostatic
interaction induced by T1317 but not in the interaction induced by eCH.
Neither compound activated the W443Y LXR
or the W457Y LXR
mutants
(data not shown).
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Models of the LXR point mutants were built based on the LXR crystal
structures to explore the structural effects of these mutations. Modeling
indicated that the W457F phenylalanine was held tightly in a conformation
similar to that of the tryptophan. When bound to T1317, the His-435 C
1
hydrogen was directed toward the
-electron cloud of the W457F
phenylalanine. However, the weaker hydrogen bond with eCH swung the His-435
C
1 hydrogen to the edge of the W457F
-electron cloud, weakening the
cation-
interaction. This rotation also brought the electropositive
N
2 hydrogen near the electropositive C
1 and C
1 hydrogens of
the phenylalanine, further opposing the interaction. Modeling suggested that
the W457Y mutant would behave similarly with respect to the cation-
interaction but that the polar hydroxyl of the tyrosine would occupy a
lipophilic pocket of LXR, destabilizing the AF2 helix and leading to the
inability of either ligand to activate this point mutant.
To rule out the potential influence of the hydrophobic portion of the LXR
agonists in these results, a hybrid molecule was synthesized in which the
epoxide of eCH was replaced by the bis-trifluoromethyl carbinol of T1317
(Fig. 1a). The T-CH
hybrid molecule was assayed for activation of the point mutated LXR-GAL4
chimeras (Fig. 5). Activation
was only observed for the W443F LXR mutant and W457F LXR
mutant,
confirming that the acidic carbinol and not the tertiary sulfonamide in T1317
was responsible for activation of the point mutants.
LXR-GAL4 chimeras were also generated in which His-421 in LXR or
His-435 in LXR
were mutated to alanine. Neither T1317, eCH, or the T-CH
hybrid could activate the H421A LXR
or H435A LXR
point mutants,
confirming the essential role of the histidine in activation of the receptor
by both the sterol and nonsterol agonists
(Fig. 5).
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DISCUSSION |
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The structures of the eCH/LXR and T1317/LXR
complexes
demonstrate that His-435 is the critical residue in the LBD that mediates
ligand activation of the receptor. All of the amino acids that line the ligand
binding pocket, including the histidine trigger and the AF2 tryptophan, are
conserved in LXR
, so the mechanism of ligand activation is almost
certainly identical. Histidine is unique among the naturally occurring amino
acids in that it is able to function as either a hydrogen bond donor or
acceptor by changing tautomers
(28). When bound to eCH,
His-435 donated a hydrogen bond to the agonist ligand, whereas it may act as
an acceptor when the acidic T1317 is bound
(Fig. 4, b and
d). Remarkably, the receptor was able to accommodate both
electrostatic states through a 1.3-Å shift in the histidine, which
permitted the perpendicular His-Trp interaction to stabilize the AF2 helix in
its active conformation in both cases. The ability of LXR to be activated by
both proton donors and acceptors suggests that the receptor may be able to
detect a range of natural ligands. It is interesting to note that although eCH
is a neutral hydrogen bond acceptor, other endogenous LXR activators
(e.g. 24(S)-hydroxycholesterol and
22(R)-hydroxycholesterol
(1,
7)) can function as both donors
and acceptors, whereas the acidic circulating cholesterol metabolite
cholestenoic acid has also been shown to activate LXR
(29). Thus, LXR shares the
property of other retinoid X receptor heterodimers such as the peroxisome
proliferator-activated receptors
(30) and pregnane X receptor
(31) that have evolved a
molecular mechanism to sense multiple lipid metabolites rather than a single
endocrine hormone.
Although we were unable to obtain diffracting apo-LXR crystals, the
structures of LXR with structurally distinct agonists suggests how the
receptor conformation may change in the absence of ligand. The hydrogen
bonding oxygen atoms of the agonists were in approximately the same location
in both structures, but His-435 shifted, depending on the strength of the
hydrogen bond. The shift of His-435 toward T1317 opened space for a water
molecule that made a hydrogen bond to the backside ND1, further stabilizing
the complex. The outward shift of His-435 in the eCH structure displaced the
water molecule and brought the backside ND1 atom closer to the carbonyl oxygen
of Ser-432, to a distance of 3.94.0 Å. In the unliganded state,
Ser-432 might form a stronger hydrogen bond with His-435, sliding the partial
negative charge of the NE2 nitrogen closer to the indole ring. This would
effectively break the cation-
interaction with Trp-457, leaving the AF2
helix-free to assume conformations that fail to stabilize coactivator
recruitment.
The eCH and T1317 ligands made contacts with residues in helices 3, 4, 5,
the -turn, helix 10, and indirectly, the AF2 helix and sat in the same
common pocket observed for other nuclear receptors. In general, the
ligand-receptor interface was dominated by hydrophobic contacts. Although a
polar interaction was observed between Glu-281 and the A-ring hydroxyl of the
sterol, no corresponding interaction was seen in the T1317 complex, suggesting
that it is not required for high affinity binding to LXR. The hydrogen bond to
the sterol A-ring hydroxyl by Glu-281, adjacent to Arg-319, is remarkably
similar to Arg-Glu pair in the estrogen receptor that binds the phenolic OH of
estradiol (17). In each case,
the hydroxyl group is bound between an arginine from helix 5 and a glutamate
from helix 3. Another important feature of both structures is that the agonist
ligands do not fill the LXR
pocket. Indeed, there was enough room for
T1317 to adopt distinct anti and gauche conformations about
the tertiary sulfonamide (Fig.
3), suggesting that the LXR pocket can potentially accommodate a
wide range of sterol and nonsterol ligands.
In conclusion, the structure of LXR in complex with eCH and T1317
identified a His-Trp switch that mediates activation of the nuclear receptor.
Sequence alignment indicates that the nuclear bile acid receptor farnesoid X
receptor has histidine and tryptophan in corresponding positions, and x-ray
crystallography suggests that it also uses a cation-
mechanism of ligand
activation (32). No other
human nuclear receptors contain tryptophan in the AF2 helix; however, several
receptors have a suitably positioned phenylalanine that could function as a
-donor. Reanalysis of the x-ray crystal structures of the vitamin D
receptor (33), thyroid hormone
receptor (34), and
retinoid-related orphan receptor
(35) shows cation-
stabilization of the AF2 helix through a His-Phe complex. Vitamin D and
thyroid hormone directly contact the histidine residue, as seen in the LXR
complexes. Since the position and electrostatic state of the histidine depends
on the hydrogen bonding character of the ligand
(Fig. 4, c and
d), it is possible that different classes of ligands
could recruit different subsets of coregulators within cells. Although we did
not detect differences in the recruitment of SRC1 peptides to LXR
or
LXR
by eCH and T1317 (Fig.
1b), the functional differences in the electrostatic
mechanism of AF2 stabilization, combined with the large ligand binding pocket,
suggest that LXR and related receptors will be a good targets for the
development of modulator ligands with improved therapeutic windows.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Present address: Pharmaceutical Product Development, Wilmington, NC
28412.
To whom correspondence should be addressed: GlaxoSmithKline, Discovery
Research, 5 Moore Dr., Research Triangle Park, NC 27709. E-mail:
shawn.p.williams{at}gsk.com.
1 The abbreviations used are: LXR, liver X receptor; eCH, 24(S),25
epoxycholesterol; LBD, ligand binding domain; NR, nuclear receptor.
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REFERENCES |
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