Hormone Selectivity in Thyroid Hormone Receptors

Richard L. Wagner, B. Russell Huber, Andrew K. Shiau1, Alex Kelly, Suzana T. Cunha Lima, Thomas S. Scanlan, James W. Apriletti, John D. Baxter, Brian L. West and Robert J. Fletterick

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Separate genes encode thyroid hormone receptor subtypes TR{alpha} (NR1A1) and TRß (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia. We previously determined the crystal structure of the rat (r) TR{alpha} ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTR{alpha} LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRß receptor LBD in a complex with either Triac or a TRß-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTR{alpha} and hTRß LBDs show close structural similarity. However, the hTRß structures extend into the DNA-binding domain and allow definition of a structural "hinge" region of only three amino acids. The two TR subtypes differ in the loop between helices 1 and 3, which could affect both ligand recognition and the effects of ligand in binding coactivators and corepressors. The two subtypes also differ in a single amino acid residue in the hormone-binding pocket, Asn (TRß) for Ser (TR{alpha}). Studies here with TRs in which the subtype-specific residue is exchanged suggest that most of the selectivity in binding derives from this amino acid difference. The flexibility of the polar region in the TRß receptor, combined with differential recognition of the chemical group at the 1-carbon position, seems to stabilize the complex with GC-1 and contribute to its ß-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors comprise one of the largest gene families, and are major targets for pharmaceuticals. These receptors mediate actions of steroid and thyroid hormones, retinoids, vitamin D, prostaglandins, fatty acids, and other regulators (1). Within the broader group are receptor subfamilies, such as the {alpha}- and ß-forms of the estrogen (ER) and thyroid hormone (TR) receptors, and {alpha}-, ß-, and {gamma}-forms of the peroxisome proliferator-activated (PPAR), retinoic acid (RAR), and retinoid-X (RXR) receptors. These receptor subtypes show extensive overlap in their hormone-binding activities. Furthermore, certain steroid receptors, such as the glucocorticoid (GR) and mineralocorticoid (MR) receptors, show subtle differences in ligand recognition. Nevertheless, as shown for the ERs, receptor subtypes can also display differential binding of certain ligands, sometimes with significant functional consequences (2, 3).

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, {alpha} and ß (NR1A1 and NR1A2) (6, 7, 8). Each gene encodes two products generated from differential RNA splicing (9). The TR{alpha}1 product represents a functional receptor and responds to thyroid hormone. The TR{alpha}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 1Goa shows the sequences of the two subtypes with the variant amino acids and ligand contacts.



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Figure 1. Thyroid Hormone Receptors and Ligands

a, Alignment of TR genes {alpha} and ß from various species. The genes for both TR subtypes from human, rat, chick, and frog were aligned using ClustalW (49 ). Consensus residues appear in normal type, variant or species-specific residues appear in reduced italic type. Positions that show a distinct subtype-specific difference (i.e. maintained in three of the four species shown here) are shaded. The {alpha}-gene consensus sequence appears in dark gray, the ß-gene consensus sequence appears in light gray. Residues that make ligand contacts, denoted by a dark sphere, are uniformly conserved across species. Only S277/N331, which indirectly contributes to ligand interaction, differs between the two subtypes. The only residue that differs between the rat and human {alpha}-genes is enclosed in a box. The conclusions here are supported by the inclusion of other species (mouse, sheep, flounder, zebrafish) in the alignment. The specific gene sequences used to construct the figure are: tha1 human (gi 135702), tha2 rat (gi 57390), tha chick (gi 135710), thaa xenla (gi 135707), thb1 human (gi 586092), thb1 rat (gi 586094), thb chick (gi 2507416) and thab xenla (gi 214831). b, Chemical structures of thyroid hormone agonists. Iodines are purple, carbons are gray, nitrogen is blue, oxygens are red, and hydrogens are white.

 
TRs contain three major domains. The amino-terminal domain (sometimes divided into the A and B domains) influences TR function in some cellular contexts (10, 11). The DNA-binding domain (DBD; also termed the C domain) anchors the receptor to specific DNA sequences. The carboxyl-terminal ligand-binding domain (LBD; sometimes referred to as the E domain) binds hormone, undergoes hormone-induced conformational changes, and interacts with various proteins such as corepressors and coactivators that mediate transcriptional effects of the receptor (12). Separating the DBD and LBD is a so-called "hinge" region (sometimes referred to as the D domain), implicated in corepressor binding and release (13). The structure of this region has not been determined for any nuclear receptor.

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{alpha}. In support of this notion, mice lacking the TR{alpha} 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. 1Go(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{alpha} and TRß LBDs in complexes with several different ligands.

We previously reported the x-ray crystallographic structure of the rat (r) TR{alpha} LBD in a complex with 3,5-dimethyl-3'-isopropyl thyronine (Dimit) (20). The structure revealed that the receptor is largely {alpha}-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{alpha} LBD structure and so reveal one structure for the D domain. For comparison, we report the structure of the rTR{alpha} in a complex with Triac at 2.5Å resolution. These results identify differences in the ligand-binding pocket between the {alpha} - and ß-subtypes, such as the role of a single amino acid residue that differs between the two (Ser 277 in TR{alpha} 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{alpha} and hTRß LBDs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Structure of hTRß with Triac
Structure Outside the Hormone-Binding Pocket.
The hTRß LBD was purified in the presence of Triac, and crystals were obtained and analyzed by x-ray diffraction as described in Materials and Methods. The structure of the hTRß LBD:Triac complex was then determined by molecular replacement (Materials and Methods) using the structure of the rTR{alpha} LBD, and refined to an R-factor of 23% (20). Table 1Go shows details of data collection and model refinement.


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Table 1. Data Collection and Refinement Statistics

 
The hTRß LBD consists of 12 {alpha}-helices and 4 ß-strands organized in three layers, as observed for the rTR{alpha} LBD (Fig. 2aGo). Consistent with the high sequence identity between the two gene products (88%), the receptors are easily superimposed [0.6Å root mean square (rms) deviation for hTRß vs. rTR{alpha} for 192 C{alpha}, as compared with 1.5 Å when superimposing hTRß vs. the human RAR-{alpha} (hRAR{alpha}) for 187 C{alpha}]. Structural differences are found in two loops that link conserved {alpha}- helices: residues A253–K263 between H1 and H3, and residues S380–L386 between H9 and H10 (Fig. 2bGo). Also, the final two turns of H11 (S437–V444) bend inward in hTRß, producing a shift in the loop between H11 and H12 (residues E445–F451). Finally, residues E202–G209, located amino-terminal to H1, form a two-turn {alpha}-helix (H0) where the analogous residues were disordered in the rTR{alpha} structure (20).



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Figure 2. Structure of the hTRß

a, Ribbon drawing of the hTRß. Secondary structure elements are labeled. The hormone, Triac, is shown in a space-filling representation. b, Superposition of hTRß and rTR{alpha}. For simplicity, secondary structures are shown as in the TRß and colored gray (rms deviation less than 0.6 Å between TR{alpha} and TRß). Differences in the position of the ß-hairpin, the loop between H9 and H10, the final two turns in H11, and the loop between H11 and H12 are labeled. The additional helix at the N terminus of the TRß, unobserved in the TR{alpha}, is also labeled. The ends of the ß-like turn between H1 and H3, unobserved in the TRß, are marked. Atoms of the TR{alpha} are in green; atoms of TRß are blue. c, Detail of the interactions of the TRß LBD with Triac. The polar region of the hormone-binding pocket (residues I276–D285 of H3, S314–Y321 of H6, and L328–V335 of the ß-hairpin turn of S3 and S4) is shown as a C{alpha} trace colored green. Individual atoms of the side chains of residues Arg282, Arg316, Arg320, and Asn331 and of Triac are colored by chemical type: oxygen, red; nitrogen, blue; carbon, green; and iodine, purple. Hydrogen bonds are indicated by black dashed lines.

 
In the TRß residues A253–K263 are disordered, the chain appearing to extend away from the receptor into the solvent. This difference in the structure of the TRß is likely forced by a crystal contact between symmetry-related molecules. If these residues were to adopt the compact conformation observed in the TR{alpha}, the neighboring molecule would clash with the folded-back structure of the loop. The same crystal contact also produces a bend in the final two turns of H11, shifting the loop between H11 and H12 (2.1Å rms). In the TR{alpha}, the H1–H3 and H11–H12 loops interact. Thus, the deviations in the loop structure between the two receptors, and increased B-factors for the H1–H3 and H11–H12 loops in the TRß (rms difference 13 Å2 after normalization, compare with 6 Å2 for H12), reflect this movement of H11 and the loss of interactions that stabilize the compact TR{alpha} conformation.

The sequence of the loop connecting H9 and H10 differs between the two subtypes (326-STDRSGLLC-334 in the TR{alpha} 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 K198–K206), 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 {alpha}-helical secondary structure. Here, the residues form a ß-like coil. The sequence of the TR{alpha} in this region, residues 155-QQRPEP-160, would also support a flexible loop.

The Hormone-Binding Pocket.
As observed in the TR{alpha}, 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{alpha}). 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{alpha} 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 2cGo 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{delta}1 of Asn331 (2.8 Å) and with the side chain of Asp285 (not shown).

Comparison of the rTR {alpha} 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{alpha} 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. 1aGo), 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{alpha} 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{alpha} 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. 3Go, 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{alpha}) differs between the two receptors. With helices H3 and H5–H6 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 H5–H6) forming a charge pair (3.4 Å) with the negatively charged acetic acid group of Triac (Fig. 3aGo). However, in rTR{alpha}, Arg228 (located on H3) rotates about the C{gamma}-C{delta} 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{alpha} (Fig. 3b).



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Figure 3. Interactions of TR{alpha} with Triac and Comparison with TRß

a, Detail of the interaction of the TR{alpha} LBD with Triac. The polar region of the hormone-binding pocket (residues I222–D231 of H3, S260–Y267 of H6, and L274–V281 of the ß-hairpin S3 and S4) is shown as a C{alpha} trace colored green. Individual atoms of the side chains of residues Arg228, Arg262, Arg266, and Ser277 and of Triac are colored by chemical type: oxygen, red; nitrogen, blue; carbon, green; and iodine, purple. Hydrogen bonds are indicated by black dashed lines. b, Comparison of TR{alpha} and TRß. Superposition of the complexes of the rTR{alpha} and hTRß with Triac. For simplicity, the positions of H3 and H6 are shown as in the TRß and colored gray (<0.3 Å rms deviation between TR{alpha} and TRß). Differences in the position of Arg228/Arg282 in H3, Ser277/Asn331, and the ß-hairpin are shown. Atoms of the TR{alpha} are in green, of TRß in blue. The torsion of Arg228/Arg282 about the C{gamma}-C{delta} is noted.

 
Structure of hTR ß 0 with GC-1
GC-1 differs from T3 in three key respects (Fig. 1bGo): replacement of the ether bridge joining the two rings with methylene; replacement of the 3, 5, and 3' iodine atoms with methyl (3, 5) and isopropyl (3') groups; and replacement of 1-amino propionic acid with oxyacetic acid. These changes to the chemical structure produce an analog with comparable dimensions to T3 with a lower molecular weight. Compared with T3, GC-1 has a greater ring separation (bond length, 1.53 Å for methylene vs.1.40 Å for ether), smaller groups at the meta positions of the phenyl rings, and a flexible 1-position substituent.

In the complex of the hTRß with GC-1, these three changes are easily accommodated in the receptor (Fig. 4aGo). 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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Figure 4. Interactions of TRß with GC-1

a, Comparison of the thyroid hormone analogs GC-1 and Dimit. Two views of the hormone analogs GC-1 and Dimit (the parent compound of GC-1) after superposition of the receptors. The outer rings superimpose, while the inner ring of GC-1 moves forward toward the polar portion of the hormone-binding pocket. Atoms are colored by chemical type: oxygen, red; nitrogen, blue; carbon, green (GC-1) or gray (Dimit). The same differences are observed relative to Triac (not shown). b, Detail of the interaction of the TRß LBD with GC-1. A ribbon drawing of part of the TRß LBD with Arg282, Arg316, Arg320, and Asn331 shows hydrogen bonds indicated by black dashed lines.

 
In the hydrophobic portion of the hormone-binding cavity, the residues adopt similar conformations to the Triac complex, with the exception of Met 310, which rotates about the C{gamma} to create a small void in the interior of the pocket. In the polar portion, Arg320 forms a hydrogen bond with the carboxylate O4 oxygen (2.8 Å). Arg316 (part of the foundation of the hormone binding pocket) interacts with the carboxylate O3 oxygen through a water-mediated hydrogen bond. Arg282 bends inward to also form a hydrogen bond to the carboxylate O3 (2.9 Å); in this conformation the N{eta} and N{epsilon} form (bifurcated) hydrogen bonds (3.1 Å and 2.7 Å, respectively) with the O{delta} of Asn 331. Additionally, the carbonyl of Leu 330 forms a close contact (3.4 Å) to the ester oxygen of the oxyacetic acid. Figure 4bGo shows the detailed interactions of GC-1 with Asn 331 and the three arginines in the polar pocket.

Role of Asn 331
As stated above, the Asn 331 in hTRß (Ser 277 in rTR{alpha}) is the only residue in the ligand-binding pocket that differs between the two subtypes (Fig. 1Go), 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{alpha} 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. 5Go) 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{alpha} 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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Figure 5. Selectivity of hTR{alpha} and hTRß for GC-1 Reversed by Swapping the Single Subtype-Specific Pocket Residue

a, Plot of Log GC-1 concentration vs. the % T3 bound for TR{alpha} LBD. In these competition ligand-binding assays, radioactive T3 was displaced from TR{alpha} LBD, WT, or mutant with Ser277 replaced with the analogous Asn from the ß-form. GC-1 binds 3-fold tighter to the Asn mutant of TR{alpha} LBD. b, Plot of Log GC-1 concentration vs. the % T3 bound for TRß-LBD. Radioactive T3 was displaced from TRß LBD, WT, or mutant with Asn331 replaced with the analogous Ser from the ß-form. GC-1 binds 3-fold weaker to the Ser mutant of TRß LBD.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the current study, we report crystallographic structures of the complex of hTRß LBD with Triac and the subtype-selective analog GC-1, and the structure of the rTR{alpha} bound to Triac. These data permit a comparative study of the two receptor subtypes. The TR{alpha} and TRß structures, while similar, show subtle differences that might translate as differences between the subtypes in regulating transcription and recognizing synthetic ligands. These differences include local changes in and around the ligand-binding pocket, and in the structure of the turn between helices 2 and 3. Unexpectedly, the crystal form for the hTRß LBD shows more order at the amino terminus than the rTR{alpha} form, providing information about the domain structure of the TR and the linkage between the DBD and the LBD.

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. 1Go(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 {alpha}-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 {alpha}-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 {alpha}-helix (residues E202–I208), which represents the C-terminal of the last TR DBD {alpha}-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 (G209–K211). 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 D104–I208) and a greater LBD (residues P214–D461), 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{alpha} and TRß: the Hormone-Binding Pocket
The LBDs of the {alpha} 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{alpha} 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. 3cGo). 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{alpha}. The backbone shift is probably induced by the position of Asn 331 in TRß (Ser 277 in TR{alpha}).

Comparison of the TR {alpha} 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{alpha}, 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 H1–H3 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 H11–H12 loop, with concomitant disruption of the H1–H3 loop (30, 31). The conformation of the H1–H3 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 H1–H3 loop. As a result of its displacement, the TRß LBD is less compact than the TR{alpha}, which could produce differences in hormone affinity, possibly through an enhanced off-rate. Furthermore, as the interactions between the H1–H3 and H11–H12 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{alpha} 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{alpha} (Fig. 5Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vectors
hTRß LBD (His6 E202-D461) was expressed from a pET28a (Novagen, Madison, WI)-based construct. The pET28 vector was cut with NdeI and BamHI, and ligated with a PstI-BamHI fragment of the TRß cDNA and an NdeI-PstI synthetic oligonucleotide encoding the TR residues upstream of the PstI site starting at E202 (7, 40). The N331S mutation was created within a TRßcDNA subclone using cassette mutagenesis, by exchanging the 96-bp region between natural BsmFI and PvuII sites for a synthetic oligonucleotide containing a Ser-encoding TCT codon at position 331. The mutant sequence was transferred to the pET28 TR-E202 vector using unique PstI and SalI sites. The sequence was verified by automated DNA sequencing at the University of California San Francisco Cancer Research Center.

hTR{alpha} 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{alpha} 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{alpha} 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 12–300 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 {alpha}-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{alpha} 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 {gamma}-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 (2–3 days, 4 C) from hanging drops (1 µl protein solution, 1 µl precipitant solution) above a reservoir containing 0.8–1.0 M sodium acetate (pH unadjusted), 50–200 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{alpha}.
Monoclinic crystals of the rTR{alpha} 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 7–1 ({lambda} = 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{alpha} 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{alpha} 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 {varsigma}-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: Glu245–Lys263. 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 Glu202–Gln252, Val264–Asp461, 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 Glu202–Gln252, Val264–Asp46, and 56 water molecules.

rTR{alpha}.
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 7–1 ({lambda} = 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 {varsigma}-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{alpha} LBD residues Arg157–Phe405; four cacodylate-modified cysteines: Cys294, Cys298, Cys388, and Cys434; and 35 solvent molecules modeled as water.


    ACKNOWLEDGMENTS
 
We thank C. A. Collins and B. D. Darimont for useful discussions and critiques of the manuscript. Figures were prepared using MOLSCRIPT (47) and RASTER3D (48).


    FOOTNOTES
 
Address requests for reprints to: Dr. Robert J. Fletterick, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143.

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. Back

Received for publication September 6, 2000. Revision received December 4, 2000. Accepted for publication December 5, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  2. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  3. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA 1998 Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139:4252–4263[Abstract/Free Full Text]
  4. Utiger RD 1995 The thyroid: physiology, thyrotoxicosis, hypothyroidism, and the painful thyroid. In: Felig PF, Baxter JD, Frohman CA (eds) Endocrinology and Metabolism. MacGraw-Hill, New York, pp 435–519
  5. von Olshausen K, Bischoff S, Kahaly G, Mohr-Kahaly S, Erbel R, Beyer J, Meyer J 1989 Cardiac arrhythmias and heart rate in hyperthyroidism. Am J Cardiol 63:930–933[Medline]
  6. Sap J, Munoz A, Damm K, Goldberg Y, Ghysdael J, Leutz A, Beug H, Vennstrom B 1986 The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature 324:635–640[Medline]
  7. Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A gene encodes a thyroid hormone receptor. Nature 324:641–646[Medline]
  8. Thompson CC, Weinberger C, Lebo R, Evans RM 1987 Identification of a novel thyroid hormone receptor expressed in the mammalian central nervous system. Science 237:1610–1614[Medline]
  9. Lazar MA 1993 Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14:184–193[Medline]
  10. Langlois MF, Zanger K, Monden T, Safer JD, Hollenberg AN, Wondisford FE 1997 A unique role of the ß-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone. Mapping of a novel amino- terminal domain important for ligand-independent activation. J Biol Chem 272:24927–24933[Abstract/Free Full Text]
  11. Safer JD, Langlois MF, Cohen R, Monden T, John-Hope D, Madura J, Hollenberg AN, Wondisford FE 1997 Isoform variable action among thyroid hormone receptor mutants provides insight into pituitary resistance to thyroid hormone. Mol Endocrinol 11:16–26[Abstract/Free Full Text]
  12. Weatherman RV, Fletterick RJ, Scanlan TS 1999 Nuclear-receptor ligands and ligand-binding domains. Annu Rev Biochem 68:559–581[CrossRef][Medline]
  13. Damm K, Evans RM 1993 Identification of a domain required for oncogenic activity and transcriptional suppression by v-erbA and thyroid-hormone receptor {alpha}. Proc Natl Acad Sci USA 90:10668–10672[Abstract]
  14. Johansson C, Vennstrom B, Thoren P 1998 Evidence that decreased heart rate in thyroid hormone receptor-{alpha}1-deficient mice is an intrinsic defect. Am J Physiol 275:R640–646
  15. Wikstrom L, Johansson C, Salto C, Barlow C, Campos Barros A, Baas F, Forrest D, Thoren P, Vennstrom B 1998 Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor {alpha} 1. EMBO J 17:455–461[Abstract/Free Full Text]
  16. Gothe S, Wang Z, Ng L, Kindblom JM, Barros AC, Ohlsson C, Vennstrom B, Forrest D 1999 Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary-thyroid axis, growth, and bone maturation. Genes Dev 13:1329–1341[Abstract/Free Full Text]
  17. Gauthier K, Chassande O, Plateroti M, Roux JP, Legrand C, Pain B, Rousset B, Weiss R, Trouillas J, Samarut J 1999 Different functions for the thyroid hormone receptors TR{alpha} and TRß in the control of thyroid hormone production and post-natal development. EMBO J 18:623–631[Abstract/Free Full Text]
  18. Chiellini G, Apriletti JW, Yoshihara H, Baxter JD, Ribeiro RC, Scanlan TS 1998 A high-affinity subtype-selective agonist ligand for the thyroid hormone receptor. Chem Biol 5:299–306[Medline]
  19. Trost SU, Swanson E, Gloss B, Wang-Iverson DB, Zhang H, Volodarsky T, Grover GJ, Baxter JD, Chiellini G, Scanlan TS, Dillmann WH 2000 The thyroid hormone receptor-ß-selective agonist GC-1 differentially affects plasma lipids and cardiac activity. Endocrinology 141:3057–3064[Abstract/Free Full Text]
  20. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  21. Clifton-Bligh RJ, de Zegher F, Wagner RL, Collingwood TN, Francois I, Van Helvoirt M, Fletterick RJ, Chatterjee VK 1998 A novel TR ß mutation (R383H) in resistance to thyroid hormone syndrome predominantly impairs corepressor release and negative transcriptional regulation. Mol Endocrinol 12:609–621[Abstract/Free Full Text]
  22. Rastinejad F, Perlmann T, Evans RM, Sigler PB 1995 Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375:203–211[CrossRef][Medline]
  23. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  24. Lin KH, Parkison C, McPhie P, Cheng SY 1991 An essential role of domain D in the hormone-binding activity of human ß 1 thyroid hormone nuclear receptor. Mol Endocrinol 5:485–492[Abstract]
  25. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-{gamma} ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  26. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-{alpha}. Nature 375:377–382[CrossRef][Medline]
  27. Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors [published erratum appears in Nat Struct Biol 1996 Feb;3(2):206]. Nat Struct Biol 3:87–94[Medline]
  28. Katz RW, Koenig RJ 1993 Nonbiased identification of DNA sequences that bind thyroid hormone receptor {alpha} 1 with high affinity. J Biol Chem 268:19392–19397[Abstract/Free Full Text]
  29. Collingwood TN, Wagner R, Matthews CH, Clifton-Bligh RJ, Gurnell M, Rajanayagam O, Agostini M, Fletterick RJ, Beck-Peccoz P, Reinhardt W, Binder G, Ranke MB, Hermus A, Hesch RD, Lazarus J, Newrick P, Parfitt V, Raggatt P, de Zegher F, Chatterjee VK 1998 A role for helix 3 of the TRß ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutations in resistance to thyroid hormone. EMBO J 17:4760–4770[Abstract/Free Full Text]
  30. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  31. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[Medline]
  32. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3343–3356[Abstract/Free Full Text]
  33. Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 280:1747–1749[Abstract/Free Full Text]
  34. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  35. Bogan AA, Cohen FE, Scanlan TS 1998 Natural ligands of nuclear receptors have conserved volumes. Nat Struct Biol 5:679–681[CrossRef][Medline]
  36. Egea PF, Mitschler A, Rochel N, Ruff M, Chambon P, Moras D 2000 Crystal structure of the human RXR{alpha} ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J 19:2592–2601[Abstract/Free Full Text]
  37. Klaholz BP, Mitschler A, Belema M, Zusi C, Moras D 2000 Enantiomer discrimination illustrated by high-resolution crystal structures of the human nuclear receptor hRAR{gamma}. Proc Natl Acad Sci USA 97:6322–6327[Abstract/Free Full Text]
  38. Jorgensen EC 1978 Thyroid hormones and analogs. II. Structure-activity relationships. In: Li CH (ed) Hormonal Peptides and Proteins. Academic Press, New York, pp 107–204
  39. Yokoyama N, Walker GN, Main AJ, Stanton JL, Morrissey MM, Boehm C, Engle A, Neubert AD, Wasvary JM, Stephan ZF, Steele RE 1995 Synthesis and structure-activity relationships of oxamic acid and acetic acid derivatives related to L-thyronine. J Med Chem 38:695–707[Medline]
  40. Beck-Peccoz P, Chatterjee VK, Chin WW, DeGroot LJ, Jameson JL, Nakamura H, Refetoff S, Usala SJ, Weintraub BD 1994 Nomenclature of thyroid hormone receptor-ß gene mutations in resistance to thyroid hormone: consensus statement from the First Workshop on Thyroid Hormone Resistance, 10–11th July 1993, Cambridge, UK. Eur J Endocrinol 130:426–428[Medline]
  41. Apriletti JW, Baxter JD, Lau KH, West BL 1995 Expression of the rat {alpha} 1 thyroid hormone receptor ligand binding domain in Escherichia coli and the use of a ligand-induced conformation change as a method for its purification to homogeneity. Protein Expr Purif 6:363–370[CrossRef][Medline]
  42. McGrath ME, Wagner RL, Apriletti JW, West BL, Ramalingam V, Baxter JD, Fletterick RJ 1994 Preliminary crystallographic studies of the ligand-binding domain of the thyroid hormone receptor complexed with triiodothyronine. J Mol Biol 237:236–239[CrossRef][Medline]
  43. Otwinowski Z, Minor W 1997 Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326
  44. Collaborative Computational Project No. 4 1994 The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50:760–763[CrossRef]
  45. Kleywegt GJ, Jones T 1994 OOPS-a-daisy. ESF/CCP4 Newsletter 30:20–24
  46. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL 1998 Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D 54:905–921[CrossRef][Medline]
  47. Kraulis P 1991 MOLSCRIPT: a program to produce both detailed and schematic plots of protein structure. J Appl Crystallogr 24:946–950[CrossRef]
  48. Merritt EA, Bacon DJ 1993 Raster3D: photorealistic molecular graphics. Methods Enzymol 277:505–524
  49. Thompson JD, Higgins DG, Gibson TJ 1994 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680[Abstract]