Department of Biochemistry and Biophysics (R.L.W., A.K.S., R.J.F.) Graduate Group in Biophysics (B.R.H., A.K.) Metabolic Research Unit, Department of Medicine (S.T.C.L., J.W.A., J.D.B., B.L.W.) Departments of Pharmaceutical Chemistry and Molecular and Cellular Pharmacology (T.S.S.) University of California, San Francisco San Francisco, California 94143
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ABSTRACT |
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INTRODUCTION |
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Thyroid hormones affect most mammalian tissues. In excess, these hormones may cause weight loss, tachycardia, atrial arrhythmias, and heart failure. Further physiological responses are reduction of plasma cholesterol levels, elevated mood, and muscle wasting (4). Some effects of thyroid hormones could be beneficial, e.g. lowering plasma cholesterol levels or inducing weight loss in obese individuals. Other effects, such as promotion of tachycardia and subsequent heart failure, are deleterious and can outweigh beneficial properties of thyroid hormone analogs (5). If hormone analogs could be made to be selective in their effects, adverse actions of thyroid hormone might be avoided.
There are two separate TR genes, and ß (NR1A1 and NR1A2) (6, 7, 8).
Each gene encodes two products generated from differential RNA splicing
(9). The TR
1 product represents a functional
receptor and responds to thyroid hormone. The
TR
2 isoform does not bind thyroid hormone but
can antagonize thyroid hormone action. The TRß1
and TRß2 isoforms differ in their amino
termini, but both bind and respond to thyroid hormone. Figure 1
a shows the sequences of the two
subtypes with the variant amino acids and ligand contacts.
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The TR subtypes can differ in their contribution to particular
responses. The finding of tachycardia in patients with elevated thyroid
hormone levels and TSH having mutations in the TRß gene (a syndrome
called resistance to thyroid hormone) suggests that the high thyroid
hormone levels might mediate this effect through normal TR. In
support of this notion, mice lacking the TR
exhibit a reduced heart
rate and an inability to generate a tachycardia, even with
administration of high doses of thyroid hormone (14, 15); conversely,
mice lacking the TRß exhibit elevated levels of TSH, suggesting a
primary role for the TRß in its suppression (16, 17).
The synthesis and characterization of a ß-subtype-selective compound,
GC-1 [3, 5-dimethyl-4-(4'- hydroy-3'-isopropylbenzyl)-phenoxy
acetic acid; Fig. 1(b)] has allowed further evaluation of the relative
contributions of the two subtypes to particular responses (18). In
hypothyroid mice, GC-1, unlike the major thyroid hormone
T3, lowers serum cholesterol at concentrations
that do not affect heart rate (19). Thus, subtype-selective hormone
analogs such as GC-1 might be employed to produce specific
thyroid hormone actions, analogous to the use of selective ER
modulators. Selective modulation of TR action might be useful in
treating obesity and hypercholesterolemia and other lipid disorders.
GC-1 has been shown to lower serum cholesterol and triglyceride
concentrations (19), risk factors for atherosclerosis. Insights into
improved subtype-selective compounds might be revealed from information
from the x-ray crystal structures of the TR
and TRß LBDs in
complexes with several different ligands.
We previously reported the x-ray crystallographic structure of the rat
(r) TR LBD in a complex with 3,5-dimethyl-3'-isopropyl thyronine
(Dimit) (20). The structure revealed that the receptor is largely
-helical with the ligand contributing part of its hydrophobic core.
In the current studies, we report structures of the hTRß LBD at 2.5Å
and 2.9Å resolution bound to the hormone analogs Triac (3,5,3'-triiodo
thyroacetic acid) and GC-1, respectively. These new structures extend
more N-terminal than the previous rTR
LBD structure and so reveal
one structure for the D domain. For comparison, we report the structure
of the rTR
in a complex with Triac at 2.5Å resolution. These
results identify differences in the ligand-binding pocket between the
- and ß-subtypes, such as the role of a single amino acid residue
that differs between the two (Ser 277 in TR
or Asn331 in TRß) that
may be useful for pharmaceutical design. The significant contribution
of this single residue in defining ß-selectivity of the binding of
GC-1 was confirmed by mutation analysis of the hTR
and hTRß
LBDs.
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RESULTS |
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The sequence of the loop connecting H9 and H10
differs between the two subtypes (326-STDRSGLLC-334 in the TR
vs. 380-SSDRPGLAC-388 in the TRß). The modest sequence
variation produces a 1.0 Å rms shift in the structure of the loop
between the two helices over the first seven of these residues. The
mutation R383H, which exhibits impaired release of corepressor,
suggests a role for the loop in this hormone-dependent activity
(21).
H0, which nestles against a neighboring TR molecule in the crystal,
contains the final four residues terminating the TR DBD (22). Assuming
the terminal helix of the TR DBD (residues K198K206), and H0 form a
continuous helix in the full-length receptor (an assumption consistent
with secondary structure prediction), the true "hinge" which links
the DBD and LBD must be residues 209-GHKPEP-214, a cluster predicted to
break -helical secondary structure. Here, the residues form a
ß-like coil. The sequence of the TR
in this region, residues
155-QQRPEP-160, would also support a flexible loop.
The Hormone-Binding Pocket.
As observed in the TR, the hormone is buried within the TRß,
providing the hydrophobic core for a subdomain of the protein. The
amino acids that form the cavity are nearly identical for the two TR
subtypes, differing only in a single amino acid residue, Asn 331
(TRß) for Ser 277 (TR
). The binding cavity may be subdivided into
two contiguous parts: a hydrophobic portion that contacts the iodinated
inner and outer rings of the hormone, and a hydrophilic portion that
interacts with the charged, polar substituent at position 1 of the
inner ring.
Interactions in the hydrophobic pocket between the TRß and Triac
largely reproduce those observed in the complex of the rTR with
Dimit (20). His 435 forms a hydrogen bond to the 4'-phenolic hydroxyl
at the far end of the pocket. Pockets for the 3- and 5-iodo
substituents are formed by the hydrophobic side chains of Ile 275 and
Ile 276 (3-iodo) and Met 310, Ala 317, Ile 353 (5-iodo), but with a
better fit than seen with the smaller methyl groups of Dimit. The
3'-iodo atom fills a pocket formed by Gly 344, Phe 269, and Met 442.
The rms deviation between Triac and Dimit after superposition of the
receptors is 0.5 Å for the atoms of the thyronine nucleus
vs. 1.8 Å for the 1-substituent, suggesting that the
smaller size of Triac is accommodated by side chain flexibility at the
polar end, rather than the hydrophobic end, of the hormone binding
pocket.
The acetic acid 1-substituent packs loosely in the hydrophilic pocket,
formed by side chains from H2, H3, H6, and S3. Fig 2c highlights
alterations in the polar region of TRß that adjust for Triac compared
with Dimit. The carboxylate group forms a compromised hydrogen
bond/electrostatic interaction (3.0 Å with poor geometry) with the
guanidinium group of Arg320 repositioned 0.5 Å toward the hormone from
that observed in the Dimit structure (Dimit is one carbon longer at the
1-position) (20). In addition, the Arg282 side chain rotates out of the
pocket, forming a hydrogen bond with the O
1 of
Asn331 (2.8 Å) and with the side chain of Asp285 (not shown).
Comparison of the rTR 0 and hTR ß 0 in Their
Complexes with Triac
To determine structural differences between the two receptors in
complexes with the same hormone, we crystallized the rTR LBD with
Triac. The extremely high sequence identity between the two genes in
the LBD (only a single amino acid difference, found at a highly
variable position, distinguishes the two species; Fig. 1a
), and
comparable ligand affinities and selectivity of the expressed LBDs
(J. W. Apriletti, unpublished data) supports the use of the
rat as a model for the human receptor. The isomorphous cocrystals with
Triac appear in the same conditions as the original rTR
crystals
containing the receptor in a complex with Dimit (20). As expected, the
two TR complexes are similar (0.3Å rms deviation for 42 C
in the
hydrophobic core). Further, the Triac-bound and Dimit-bound TR LBDs are
nearly identical outside of the ligand-binding pocket.
In the hormone-binding pocket, few differences distinguish between the
two receptors (Fig. 3, a and b). In the
hydrophobic region, the conformations are virtually identical, to the
level of side chain rotamers. In the polar pocket, however, the
position of a structural element of the LBD, the ß-hairpin between S3
and S4 (residues Asn 331 to Glu 333 of hTRß, residues Ser 277 to Glu
279 of rTR
) differs between the two receptors. With helices H3 and
H5H6 providing a static reference for comparison, the ß-hairpin in
TRß shifts 0.9 Å closer to the carboxylate of Triac. The backbone
shift in TRß is probably induced by the position of Asn 331, the
single variant amino acid in the vicinity of Triac. Surprisingly, most
of the residues in the polar pocket of both structures adjust to adopt
the same conformations and make the same interactions with the ligand,
with Arg266 (Arg320 in the hTRß; helix H5H6) forming a charge pair
(3.4 Å) with the negatively charged acetic acid group of Triac (Fig. 3a
). However, in rTR
, Arg228 (located on H3) rotates about the
C
-C
bond toward the ligand as observed in the Dimit structure and
forms a hydrogen bond to Ser277 (2.8 Å). In the hTRß, the analogous
residue Arg282 points away from the ligand. The alternate conformation
of Arg228/282 results from both structural and sequence differences
between the subtypes. In the hTRß, the repositioning of the
ß-hairpin places the larger side chain Asn331 in the way, precluding
the inward conformation of Arg282 adopted in TR
(Fig.
3b).
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In the complex of the hTRß with GC-1, these three changes are easily
accommodated in the receptor (Fig. 4a).
When superimposed on the Triac complex, the outer rings overlap, with
the hydrogen bond to His 435 maintained and the 3' isopropyl group
occupying the same pocket as the 3'-iodine. The torsion angle of the
two rings differs by 17 degrees, due to the stereochemistry of the
methylene bridge. The inner ring lies closer to the polar pocket (0.3
Å shift) as a consequence of the longer bridge. The shift toward the
polar pocket positions the 3 and 5 methyl groups slightly forward of
the iodine, but the smaller volume of the methyl prevents a steric
clash with the residues defining the pockets.
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Role of Asn 331
As stated above, the Asn 331 in hTRß (Ser 277 in rTR) is the
only residue in the ligand-binding pocket that differs between the two
subtypes (Fig. 1
), and this residue forms hydrogen bonds in the polar
part of the hormone-binding pocket. Is this new interaction promoting
the affinity of GC-1 for the hTRß? If Asn331 stabilizes the hTRß
complex with GC-1 by forming hydrogen bonds to Arg282, accounting for
its preference for that subtype, then changing this Asn to a Ser might
produce a receptor that behaves more like the ß-subtype. To test the
importance of this difference, a variant hTRß was constructed by
substituting a serine residue for asparagine 331 (designated N 331 S),
and a reciprocal variant hTR
was constructed by substituting an
asparagine residue for Ser 277 (designated S 277 N). Hormone binding
assays were performed by competing
[125I]T3 from each of
these receptors and receptor variants with GC-1. A comparison of the
Ki values calculated for the GC-1 competitions
(Fig. 5
) show that in the TRß a serine
cannot fulfill the role of the asparagine in binding GC-1, and the
apparent binding affinity weakens to 194 pM. Reciprocally,
in TR
the substitution of the asparagine for the natural serine
strengthens the apparent binding affinity to 64 pM. It is
likely that the Asn is a major discriminator for the added affinity of
the GC-1 compound for wild-type (WT) TRß.
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DISCUSSION |
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Domain Structure of the TR: Definition of a Structural Hinge
The modular structure of nuclear receptors was inferred from
clusters of primary sequence conservation. Analysis of truncated
receptors demonstrated that two of these sequence domains, the C and E
domains, could fulfill specific activities of the intact receptors, DNA
binding and hormone binding, respectively. The D domain, separating the
C and E domains, was defined based on low sequence conservation across
the nuclear receptor family (e.g. GR vs. TR),
although significant conservation can occur in a particular receptor
across species [e.g. Xenopus (x) TRß
vs. hTRß, Fig. 1(a)]. It was suggested that the D domain
performed a role as a connection between the DBD and LBD, as a hinge
(23). Therefore, it was surprising that residues within the D domain
were required for hormone binding (24). However, the first structures
of nuclear receptor LBDs showed that some of the residues within the
hinge domain fold as part of the LBD, forming
-helix H1 and
contributing to the hydrophobic core (20, 25, 26). A reexamination of
the nuclear receptor gene family based on the LBD structure uncovered a
hydrophobic sequence signature in the D region of other receptors,
which corresponds to H1 in the LBD fold (27). Thus, part of the
region considered a hinge formed part of the structure of the
LBD.
In the case of the TR, efficient DNA binding also required residues
within the D domain. Structurally, these residues form a turn after the
second helix of the DBD (A-box), which participates in formation of
heterodimers of the TR with RXR, and an extended carboxyl-terminal
-helix, which binds in the minor groove of the DNA to allow binding
to an octamer half-site (22, 28).
In the hTRß structure, we observed a short -helix (residues
E202I208), which represents the C-terminal of the last TR DBD
-helix. Thus, our TRß structure contains the C-terminal end of
the DBD, connecting peptides and H1 of the LBD. This actual linkage
between the two domains contains only three residues (G209K211).
Thus, the D domain does not exist as a domain in either a structural or
functional sense: what was previously termed the D domain is instead
divided between a greater DBD (residues D104I208) and a greater LBD
(residues P214D461), connected by a few amino acids. We suggest that
future references to residues within the D domain instead be described
as residing in either the DBD or the LBD, to accurately reflect the
structural organization of the TR.
Comparison of the TR and TRß: the Hormone-Binding Pocket
The LBDs of the and ß TRs are markedly conserved in residues
that contact ligand and show only one amino acid residue difference
around the hormone-binding pocket. This is in contrast to the case with
the different subtypes of the RARs or the PPARs, and other nuclear
receptor gene families (25). Given that strong sequence conservation
may reflect evolutionary pressure, acting to maintain an optimal
configuration of the pocket (and the remarkable fit between the
receptor and hormone), the existence of the Ser 277/Asn 331 subtype
difference is notable. However, it limits the options for
pharmaceutical design of subtype-selective compounds based only on
differences in residues lining the pocket.
Data from the current studies allowed us to compare the
differences in this pocket between the rTR and hTRß in their
complexes with Triac. The conformations were found to be virtually
identical in the hydrophobic region, to the level of side chain
rotamers. Most of the residues in the polar pocket of both structures
adopt the same conformations and interactions with the ligand, with
Arg266 (Arg320 in TRß) forming a charge pair with the negatively
charged acetic acid group of Triac (Fig. 3c
). However, in the polar
pocket, the LBD ß-hairpin comprised of S3 and S4 is shifted in TRß
closer to the carboxylate of Triac than in the TR
. The backbone
shift is probably induced by the position of Asn 331 in TRß (Ser 277
in TR
).
Comparison of the TR 0 and TRß: Differences in the Loop
between Helices 2 and 3
The current studies revealed a second major difference between the
two subtypes that was outside the ligand-binding pocket in the
structure of residues in the loop between H1 and H3. These residues are
ordered in the TR, forming a reverse turn with ß-sheet character,
but are disordered in the TRß. The structure of the corresponding
region in the unliganded RXR was proposed to undergo a reorientation
upon hormone binding (26). In the hormone-bound TR, several residues in
the H1H3 loop form van der Waals contacts with hydrophobic residues
in the loop between H11 and H12, and mutation of these residues in
either loop produces hormone resistance (29). Furthermore, comparisons
of structures of the ER LBD with agonists and antagonists (estradiol
vs. raloxifene; DES vs. tamoxifen) show
plasticity in H12 and the H11H12 loop, with concomitant disruption of
the H1H3 loop (30, 31). The conformation of the H1H3 loop is thus
affected by the presence and nature of the hormone and directly
influences the position of H12, part of the coactivator interaction
surface (32, 33, 34).
The structural difference between the TR subtypes demonstrates the
mobility of the H1H3 loop. As a result of its displacement, the TRß
LBD is less compact than the TR, which could produce differences in
hormone affinity, possibly through an enhanced off-rate. Furthermore,
as the interactions between the H1H3 and H11H12 loops reinforce
through packing, and both are important to proper orientation of H12
for interaction with coactivators, the structural difference observed
here might indicate the ß-subtype could be more susceptible to
displacement of H12 by a hormone antagonist.
Design and Therapeutic Potential
Selective modulation of thyroid hormone action may have medical
importance in treating conditions such as hypercholesterolemia,
hypertriglyceridemia, and obesity. The finding that the TRß-selective
compound GC-1 can lower cholesterol and triglyceride levels in mice
without adversely affecting the heart rate supports the potential
utility of this class of compounds. Therefore, it is of importance to
understand in detail the basis for generating ß-selectivity (18).
Several factors appear to contribute to discrimination of various ligands for nuclear receptors. Agonists that bind to nuclear receptors fall within a small range of size, since the hormone occupies a large proportion of the hydrophobic core of the receptor (35). Thus, the shape of the ligand-binding pocket is generally important for ligand discrimination and is the dominant factor in the discrimination between retinoid isomers by the RAR and RXR retinoid receptors (36). However, with some receptors, including the TR, the shape of the ligand-binding pocket is similar between receptor subtypes. Subtle differences between specific receptor side chains can produce discrimination between closely related ligands at the level of a single atom (37). Differences in flexibility within a protein could also contribute to discrimination between closely related nuclear receptors. For example, with nuclear receptors, flexibility is required to permit entry and exit of the ligand from its internal binding site, and for formation of surfaces for binding divergent molecules such as DNA, coactivators, and corepressors. Adaptation of the receptor to distinct molecular shapes has been observed in the ER and is especially important for receptor binding of antagonists (30, 31).
The structures of the rTR and hTRß in complexes with Triac and of
the hTRß with bound GC-1, plus our examination of mutated TRs,
provide information about generation of ß-selectivity. Conformational
differences between the two subtypes when bound with Triac are subtle:
a relative displacement of the ß-hairpin of the hTRß LBD toward the
1-position of the hormone. However, in the GC-1 complex, the same
reorientation of the ß-hairpin permits the interaction of Asn331 with
Arg282, presumably stabilizing the latter in its hydrogen bond to that
analog. Thus, the selectivity of GC-1 in binding the TRß could be due
to the presence of the asparagine rather than serine with different
hydrogen bonding potential, and the flexibility of the hairpin loop.
The quantitative importance of the presence of Asn 331 instead of Ser
in the hTRß was tested by examining the binding of a hTRß in which
Ser was inserted for Asn at 331. This substitution produced a receptor
with an affinity for GC-1 that was nearly as poor as that for the
hTR
(Fig. 5
). Therefore, most of the difference in affinity is
specifically due to the single amino acid substitution.
Another feature of the GC-1 complex may be informative for design of selective ligands. Early studies established the substituent preference for the TR at the 3 and 5 positions of thyroid hormone as I > Br > methyl (38). By that formulation, GC-1 should exhibit a markedly reduced affinity for the receptor, yet GC-1 binds the ß-subtype with an affinity that is comparable to that of T3. A similar observation was noted for another series of thyroid hormone analogs with oxamic acid derivatives at the 1-position (39). The structure of the GC-1 complex suggests that the smaller methyl groups can increase rather than decrease the ability of the compound to bind to the receptor, since they allow for adjustment of the inner ring to permit optimal hydrogen bonding. Modeling suggests that the larger iodine atoms would lead to a steric clash. Thus, rather than considering the 3, 5, and 1 substituents independent in their contribution to ligand affinity, they should be regarded as coupled.
In contrast to the notion that hydrogen bonds provide specificity, the polar region of the pocket seems flexible, and less discriminating than the hydrophobic part. The flexibility allows the formation of additional hydrogen bonds between Asn331 and Arg282. The results clearly support a design program that varies the chemical groups at the 1-position of the inner ring, while preserving the negative charge, but that incorporates substituents for the hydrophobic portion that permits the design features at the 1-position to function.
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MATERIALS AND METHODS |
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hTR LBD (His6 E148-V410) was also expressed
from the pET28a vector. The pET28 vector was cut with NdeI
and EcoRI, and ligated with a
BsmFI-EcoRI fragment of the TR
cDNA and an
NdeI-BsmFI synthetic oligonucleotide encoding the TR
residues upstream of the BsmFI site starting at E148. The
S277N mutation was created within the pET28 hTR
LBD vector using the
Quick-change method with Pfu polymerase (Stratagene, La
Jolla, CA).
Protein Expression
hTRß LBD was expressed in BL21DE3 (14 C, 1 mM
isopropylthiogalactoside added at OD600 = 0.7,
induced 24 h). WT and mutant receptors for ligand-binding studies
were expressed from the pET28 TR LBD plasmids using TNT T7 Quick
in vitro translation kits (Promega Corp.,
Madison, WI).
Protein Purification
For TRß LBD, 50 mM sodium-phosphate, pH 8.0, 300
mM NaCl, 10% glycerol, 25 mM
ß-mercaptoethanol, 0.1 mM phenylmethylsulfonylfluoride
was used to lyse the cells. The steps in purification were as follows:
freeze-thaw, incubate with 0.1 mg/ml lysozyme (20 min, 0 C); sonicate,
clear lysate (Ti45, 36,000 rpm, 1 h, 4 C). Load lysate on Talon
resin (CLONTECH Laboratories, Inc., Palo Alto, CA)
equilibrated in sodium phosphate buffer, eluted with 12300
mM imidazole gradient. Isolation of liganded receptor using
TSK-phenyl HPLC (TosoHaas, Philadelphia, PA) was performed as described
previously (41). The ligands used included Triac (Sigma,
St. Louis, MO) or GC-1 (18). The overall yield was 9.5 mg/liter
bacterial culture. For crystallization, hTRß LBD was diluted into 20
mM HEPES, pH 7.4, 3 mM
dithiothreitol (DTT), and 0.1 µM appropriate
ligand using NAP columns (Pharmacia Biotech,
Piscataway, NJ), and concentrated to 9 mg/ml by ultrafiltration
(UFV2BGC10, Millipore Corp., Bedford, MA ).
The rat -isoform of the TR LBD (Met122 to
Val410), was purified as described previously
(41).
Ligand-Binding Assays
The affinities of binding of T3 to the
hTR LBD (WT and S277N mutant), and the hTRß LBD (WT and N331S
mutant) were determined using saturation binding assays. Fifteen
femtomoles of each in vitro translated receptor were
incubated overnight at 4 C with varying concentrations of
L-3,5,3'-[125I]T3
(NEN Life Science Products, Boston, MA) in a 100 µl
volume of E400 buffer (400 mM NaCl, 20
mM KPO4, pH 8, 0.5
mM EDTA, 1.0 mM
MgCl2, 10% glycerol) and also containing 1
mM monothioglycerol and 50 µg calf thymus
histones (Calbiochem, La Jolla, CA). The receptor-bound
[125I]T3 was isolated by
gravity flow through a 2 ml course Sephadex G25 (Pharmacia Biotech) column and quantified using a
-counter (COBRA,
Packard Instruments, Meriden, CT). Binding curves were fit by nonlinear
regression and the Kd values were calculated
using the one-site saturation binding model contained in the Prism 3.0
program (GraphPad Software, Inc., San Diego, CA).
The GC-1 competition assays for each LBD were similarly performed
except each incubation was 400 µl and contained 0.5
nM [125I]
T3 and varying concentrations of GC-1. The
Ki values of GC-1 for each receptor LBD were
calculated using the one-site competition model contained in the Prism
3.0 program, using the dissociation constant (Kd)
values for T3 binding to each receptor obtained
from the previous
[125I]T3 saturation
experiments.
Crystallization Trials
hTRß.
Initial crystallization conditions for hTRß LBD in a complex with
Triac were identified using Hampton Crystal Screens (Hampton Research,
Laguna Niguel, CA). A single crystal appeared (12 h, 25 C) by
hanging-drop vapor diffusion from a drop (1 µl of TRß LBD solution
9 mg/ml, 1 µl precipitant solution) suspended above a reservoir
composed of 1.4 M sodium acetate
(NaH3OAc) and 0.1 M sodium cacodylate
(NaCac), pH 6.5, at (Hampton condition no. 7). Refinement of the
conditions (1.0 M NaOAc, 100 mM NaCac, pH 7.2)
results in hexagonal bipyramidal crystals of dimensions 0.2 mm x
0.2 mm x 0.6 mm at 4 C. Crystals were space group P3121
(a = 68.9Å, c = 131.5Å) and contained 1 molecule of TRß
LBD. The N-terminal His-tag was not removed before crystallization.
Crystals of the hTRß LBD with bound GC-1 are grown (23 days, 4 C) from hanging drops (1 µl protein solution, 1 µl precipitant solution) above a reservoir containing 0.81.0 M sodium acetate (pH unadjusted), 50200 mM sodium succinate, and 0.1 M sodium cacodylate (pH 7.2). The best crystals have a smallest dimension of 200 µM. The unit cell dimensions are cell length a = 68.73Å, c = 130.09.
rTR.
Monoclinic crystals of the rTR LBD are grown using hanging-drop
vapor diffusion at ambient temperature (17 C to 22 C), from drops
containing a 2:1 mixture of approximately 10 mg/ml protein, in 20
mM HEPES, pH 7.4, and 3 mM DTT, and a reservoir
solution of 15% 2-methyl-2,4-pentanediol (MPD), 0.2 M
ammonium acetate (NH4OAc), buffered by 0.1
M sodium cacodylate at pH 6.7 (20, 42). The crystals grow
in the presence of a fluffy, white precipitate. The space group is C2
with cell lengths a = 117.16 Å, b = 80.52 Å, c = 63.21
Å, ß =120.58.
Structural Analysis
hTRß.
Crystals (Triac) were transferred briefly into a cryoprotectant
composed of 30% glycerol, 1.4 M NaAc, and 100
mM NaCac, pH 7.2, and then suspended in a nylon loop
attached to a mounting pin and flash frozen (liquid nitrogen). Crystals
(GC-1) are transferred first into cryoprotectant composed of 15%
glycerol, 1.2 M NaAc, 0.1 M NaCac, followed by
a second transfer into an identical solution at 30% glycerol, and then
flash frozen.
Diffraction data (Triac) were measured to 2.4 Å using synchrotron
radiation at Stanford Synchrotron Radiation Laboratory beamline 71
( = 1.08Å); reflections were recorded using a MAR image
plate detector and integrated with Denzo, with equivalent reflections
scaled using Scalepack (43). Data (GC-1) were measured at UCSF using Cu
K
radiation (R-axis generator, 50 kV, 300 mA, 0.3-mm collimator, Ni
filter); reflections were recorded using an R-axis image
plate area detector.
A molecular replacement solution for the hTRß LBD/Triac complex using
AMORE (CCP4, http://www.dl.ac.uk/CCP/CCP4) from the model of the
rTR LBD, with ligand omitted (44). Strong peaks are obtained in both
the rotation and translation searches, with no significant (>0.5 times
the top peak) false solutions; strong positive electron density for the
iodine atoms in both the anomalous scattering and conventional
difference electron density maps confirmed the solution. Initial maps
are calculated using
-A-weighted coefficients output by REFMAC
(CCP4, http://www.dl.ac.uk/CCP/CCP4) after nine cycles of
maximum likelihood refinement (44). The real-space fit for each residue
was calculated using OOPS (xray.bmc.uu.se) (45), and the residues with
a real-space fit less than 2 SD below the mean were
removed: Glu245Lys263. To reduce bias, the following residues were
modeled as alanine: Arg282, Arg316, Arg320, Asn331. Cycles of
rebuilding and positional least squares, simulated annealing, and
restrained, grouped B factor refinement with XPLOR (xplor.csb.yale.edu)
produce a model with an Rcryst of 20.1% and an Rfree of 24.3%. The
final model consists of hTRß LBD residues Glu202Gln252,
Val264Asp461, and 45 solvent molecules modeled as water.
The TRß LBD complex with GC-1 was refined against a maximum likelihood target using CNS (46). To reduce bias, the following residues were modeled as alanine: Arg282, Arg316, Arg320, Asn331. The true side chain was fit to difference electron density after simulated annealing. A model of the GC-1 ligand was refined or relaxed using the AMBER force field (Oxford Molecular, Inc., Palo Alto, CA) and fit to difference electron density. Refinement included positional refinement with CNS interspersed with manual rebuilding. A flat bulk solvent correction and an overall anisotropic B-factor correction were applied. The refined model consists of residues Glu202Gln252, Val264Asp46, and 56 water molecules.
rTR.
Crystals may be frozen in a nitrogen stream directly from the mother
liquor. Data were measured to 2.5 Å using synchrotron radiation at
Stanford Synchrotron Radiation Laboratory, beamline 71 ( =
1.08Å); reflections were recorded using a MAR image plate detector and
integrated with Denzo, with equivalent reflections scaled using
Scalepack (43).
Initial maps are calculated using -A weighted coefficients output by
REFMAC after nine cycles of maximum likelihood refinement (44). To
reduce bias, the following residues were modeled as alanine: Arg228,
Arg262, Arg266, Ser277. Cycles of rebuilding and positional least
squares, simulated annealing, and restrained, grouped B factor
refinement with XPLOR produce a model with an Rcryst of 19.6% and an
Rfree of 25.2%. The final model consists of rTR
LBD residues
Arg157Phe405; four cacodylate-modified cysteines: Cys294,
Cys298, Cys388, and Cys434; and 35 solvent molecules modeled as
water.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the NIH (Grant DK-53417 to R.J.F. and Grant DK-41842 to J.D.B.). S.T.C.L. was the recipient of a fellowship funded by FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo).
Dr. J. D. Baxter has proprietary interests in, and serves as a consultant and Deputy Director to Karo Bio AB, which has commercial interests in this area of research.
Coordinates are available immediately from the Protein Data Bank and from the Fletterick lab home page (http://msg. ucsf.edu/flett/).
1 Present address: Tularik Inc., South San Francisco California
94143-0448.
Received for publication September 6, 2000. Revision received December 4, 2000. Accepted for publication December 5, 2000.
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REFERENCES |
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