Two Resistance to Thyroid Hormone Mutants with Impaired Hormone Binding

B. Russell Huber, Ben Sandler, Brian L. West, Suzana T. Cunha Lima, Hoa T. Nguyen, James W. Apriletti, John D. Baxter1 and Robert J. Fletterick

Graduate Group in Biophysics (B.R.H.), Department of Biochemistry and Biophysics (B.S., R.J.F.), and Diabetes Center and the Metabolic Research Unit, Department of Medicine (B.L.W., S.T.C.L., H.T.N., J.W.A., J.D.B.), University of California, San Francisco, San Francisco, California 94143-0448

Address all correspondence and requests for reprints to: R. J. Fletterick, University of California, San Francisco, Department of Biochemistry Biophysics, 513 Parnassus Avenue, San Francisco, California 94143-0448. E-mail: flett{at}msg.ucsf.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Resistance to hormones is commonly due to mutations in genes encoding receptors. Resistance to thyroid hormone is due mostly to mutations of the ß-form of the human (h) thyroid hormone receptor (hTRß). We determined x-ray crystal structures of two hTRß ligand-binding domains (LBDs), Ala 317 Thr and Arg 316 His. Amino acids 316 and 317 form part of the hormone-binding pocket. The methyl of Ala 317, contacting iodine, sculpts the T3 hormone-binding pocket. Arg 316 is not in direct contact with T3 and has an unknown role in function. Remarkably, the Arg forms part of an unusual buried polar cluster in hTRß. Although the identity of the amino acids changes, the polar cluster appears in all nuclear receptors. In spite of the differing roles of 316 and 317, both resistance to thyroid hormone mutants display decreased T3 affinity and weakened transcriptional activation. The two mutants differ in that the Arg 316 His receptor does not form TR-TR homodimers on DNA. 3,5,3'-Triiodothyroacetic acid is bound to both receptors. Thr 317 repositions 3,5,3'-triiodothyroacetic acid distending the face of the receptor that binds coregulators. Arg 316 forms two hydrogen bonds with helix 1. Both are lost with mutation to His displacing helix 1 of the LBD and disordering the loop after helix 1. The stability of the helix 1, deriving in part from the buried polar cluster, is important for hormone binding and formation of TR dimers. The observation that the Arg 316 His mutation affects these functions implies a role for helix 1 in linking hormone binding to the DNA-binding domain-LBD configuration.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CLASSICAL ENDOCRINE DISEASES reflect hormone excess or deficiency, but there is growing focus on alterations in sensitivity to hormones in disease, including hormone resistance. A number of resistance syndromes involve mutations in nuclear receptor proteins, such as those responsive to androgens (1), glucocorticoids (2), mineralocorticoids (3), vitamin D (4), peroxisomal proliferator-activated receptor (5), and thyroid hormones [resistance to thyroid hormone (RTH; Ref. 6). These syndromes generally have in common high levels of the cognate hormone with decreased hormone response in target tissues.

RTH, reported in more than 700 humans, is caused mostly by mutations in the thyroid hormone receptor-ß (TRß) isoform gene. The syndrome is autosomal dominant, as the abnormal receptors interfere with actions of the normal receptors. Most mutated receptors have impaired binding of T3. In many cases, RTH is associated with higher levels of the thyroid hormones, T4 and T3. Normally, T4 and T3 levels are regulated by TSH, which stimulates hormone production by the thyroid gland; in turn, TSH levels are suppressed by T3 through a negative feedback mechanism. The higher levels of T3, when they occur, are due to heightened TSH levels; in many RTH cases, the higher levels of hormone compensate for the defect, leading to a mostly normal state, but when this does not occur the disease can be serious.

All mutations of human (h)TRß defined to date are in the receptor’s ligand-binding domain (LBD). As with nuclear receptors in general, binding of hormone induces folding of the carboxyl-terminal helix 12 into the body of the LBD. This results in dissociation of a corepressor that in part occupies the groove into which helix 12 fits and in formation of a surface that binds coactivators. Depending on the DNA site, the TRß may change its dimer partner. Hormone binding also stabilizes the amino-terminal region of the LBD (7). Dynamic flexibility in this region has also been noted in an nuclear magnetic resonance (NMR) structure of the unliganded peroxisomal-activated receptor LBD (8).

An atomic model of hTRß LBD from the x-ray crystallographic structure of the rat TR{alpha} LBD (9), and subsequently a structure of the hTRß LBD (10) provided insights for dysfunction for many of the mutants. The mutations cluster in three separate regions of the hTRß LBD amino acid (11, 12, 13, 14), but in three dimensions most are revealed to be around the T3 binding pocket of the receptor (15). The proximity of the mutations to the pocket explains defective binding of hormone. However, the observation that the mutations are located in different regions relative to the pocket may also explain why the mutated receptors differ from each other in other receptor functions such as interactions with corepressors, coactivators, and configuration of the receptor dimer bound to DNA. Structures of selected mutants may provide mechanistic details for the nature of the dysfunction and reveal critical components of function in addition to helix 12 and the hormone pocket.

We report x-ray crystal structures of LBDs of RTH TR mutants, Arg 316 (12, 16) and Ala 317 Thr (Fig. 1Go) (12), which occur commonly in the syndrome. These mutations are similar in that they both show significant impairment in T3 binding. Clinically, RTH arising from either Arg 316 His or Ala 317 Thr presents with high serum levels of T3, goiter, and tachycardia, but the patients are otherwise euthyroid. In cell cultures the defects in function for these mutants can be partly compensated by providing increased concentrations of T3. Like most RTH mutants, Ala 317 Thr receptors can form both homodimers and heterodimers with retinoid X receptors. The Arg 316 His mutant is unique in that only heterodimers can form (17).



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Figure 1. Thyroid Hormone Receptor RTH Mutations

In this schematic the structural elements of the native hTRß LBD are displayed in ribbon representation colored from blue to orange as the chain proceeds from the N to the C terminus. The {alpha}-helix in magenta is the glucocorticoid receptor-interacting protein nuclear receptor 2 peptide binding at the coactivator site above helix 12. A small region of ß-sheet observed in residues 327–336, known as the ß-hairpin, is colored green. Locations of the RTH mutations, Ala 317 and Arg 316 carbon C{alpha}’s and side chains are shown in gray space-filling representation, just above the hormone T3, which is presented in stick representation. This figure was composed with Swiss PDB Viewer using the structural coordinates of the hTRß bound to the glucocorticoid receptor interacting protein nuclear receptor 2 peptide (Protein Data Bank accession no. 1BSX) presented by Darimont et al. (29 ).

 
The three-dimensional structures show two different mechanisms of impaired hormone binding and identify other receptor features that may explain the difference in function of the Arg 316 His receptor. With Arg 316 His, key hydrogen bonds to helix 1 are unable to form, disrupting a buried polar cluster of side chains and the stability of helix 1. T3 moves in Ala 317 Thr because the side chain of the altered amino acid impinges on the pocket into which hormone T3 fits. In both cases, movements of up to 2 Å of amino acids around helix 1 are found. The structure of Arg 316 His also reveals the importance of interactions of helix 1 with the body of the receptor for hormone binding and formation of TR-TR homodimers. Thus, the studies suggest distinct mechanisms whereby hormone binding can be altered and provide rational explanations as to the differential behavior of mutated TRs in RTH.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Crystals of the purified Ala 317 Thr and Arg 316 His hTRß LBDs with 3,5,3'-triiodothyroacetic acid (Triac) were obtained and analyzed by x-ray diffraction (Materials and Methods). Triac was found to be the hormone promoting the highest quality crystals of several tested. Statistics from x-ray diffraction data measurement and coordinate refinement for these mutants are summarized in Table 1Go. Electron density maps of the mutation sites were calculated for both mutants with the mutant residues and surrounding structural elements removed to minimize bias in constructing the atomic coordinates for the LBDs. The experimental electron density around the mutation sites clearly defines the new positions of the mutated amino acid side chains.


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Table 1. Data Collection and Refinement Statistics

 
The structure of the native hTRß LBD is little changed by the mutations with an overall root mean square deviation (RMSD), of positional change in atomic coordinates of 0.56 Å for Ala 317 Thr and 0.62 Å for Arg 316 His. Experimental positional error is expected to be about 0.3 Å. The crystals of Arg 316 His diffract more weakly (3.1 Å) than Ala 317 Thr (2.4 Å). As observed for all nuclear receptor family members, the fold is composed primarily of 12 helices in a three-layer {alpha}-helical sandwich. Both structures also reveal helix 0, the C terminus of the DNA-binding domain (DBD) that is connected to helix 1 of the LBD by a strand of five amino acids.

The Ala 317 Thr Mutation Repositions the Hormone in the Binding Pocket
Ala 317 is located in the interior of the hTRß T3 binding pocket. Its mutation to Thr replaces two hydrogens with a hydroxyl and a methyl group. Like Ala 317, the side chain of Thr 317 contacts the iodine at position 3 of Triac as seen in Fig. 2Go. The closest contact distance between iodine and Thr 317 is 3.6 Å, compared with 4.1 Å for Ala 317. Relative to the native LBD, in Ala 317 Thr, the center of mass of the hormone moves away from the site of mutation by 0.3 Å. The contacts between the hormone and the receptor observed in wild type TRß (10) are conserved. However, the hormone binding pocket is deformed by about 0.3–0.5 Å on the side opposite the mutation. Thus, close contact by the mutated amino acid of Triac in the hormone binding pocket, with distortion of the pocket, can explain why this mutant LBD has a lower affinity for T3 relative to the native LBD.



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Figure 2. Comparison of the Ala 317 Thr T3 Binding Pocket with that of the Native Receptor

In this schematic, a slice through the hormone-binding pocket, in space filling representation illustrates slight shift of Triac to the right caused by the side chain of Thr 317. The synthetic hormone Triac is colored purple. Ala 317 is green in the native structure: Thr 317 is dark orange. Other atoms lining the pocket are gray.

 
Repositioning of the Hormone in the Ala 317 Thr Mutation Induces Packing Defects in the Receptor Opposite the Mutation
The volume of the Ala 317 Thr T3 receptor pocket is identical within experimental error to the binding pocket of the native hTRß LBD, being smaller by approximately 10 Å3 of about 600 Å3. In contrast, the overall volume of the Ala 317 Thr receptor LBD is increased by about 300 Å3 relative to the native hTRß LBD as a result of the disrupted packing. This volume is too small to visualize as it is equivalent to only about 10 methyl groups distributed throughout the 30,000 Å3 of the entire LBD. Helix 6, which contains residue 317, and helices of the receptor hydrophobic core remain unmoved. Instead, expansion of the LBD occurred primarily on the side of the receptor opposite the mutation site, with movements of helix 3 of approximately 1 Å, the loop between helix 1 and helix 2 of about 1 Å, the ß-hairpin of about 1 Å, residues 327–335. Thus, through collisions with the Triac, or T3, a mutation that has a minimal steric compromise in the hormone binding pocket moves the face of the receptor in the helix 1–2 region out, suggesting that this portion of the LBD is less well packed and more dynamic than the opposite side of the LBD.

The Arg 316 His Mutation Disrupts a Polar Cluster
Arg 316 is remarkable for a charged residue in that it is buried inside the protein and inaccessible to solvent. In the native structure Arg makes three apparent hydrogen bonds in a network of four buried polar side chains plus hormone. One of these hydrogen bonds is with the solvent-inaccessible Gln 374 on helix 9. The other two hydrogen bonds are with Thr 232, on helix 1, the linkage to the DBD. These polar interactions probably balance the full positive charge expected to be on the Arg guanidinium nitrogen atoms, permitting a stable configuration for the buried Arg. One likely function of Arg 316 is to link helix 1 to the body of the receptor via hydrogen bonds to helix 1 and helix 9, as shown in Fig. 3Go. Mutation of Arg 316 to His changes the hydrogen bonding configuration and electrostatic charge of the side chain. In the mutant structure the side chain of His 316 forms a single hydrogen bond with Gln 374 on helix 9, breaking the connection between helix 1 and helix 6. Helix 1 is displaced away from the hormone and the body of the receptor with an RMSD of about 1.0 Å: a small portion of helix 3, adjacent to helix 1, moves similarly by an RMSD of 0.8 Å away from its native position. These concerted movements of the surface helices permit not-so-subtle relaxation of the underlying side chains on which the helices rest. Of several large-scale rearrangements under helix 1, notable are Asp 85 and Phe 86, which move in the direction of helix 1 by more than 1.0 Å, and Phe 187, which moves by about 2.0 Å. The position of the hormone within the pocket is unchanged, but the alterations of the underlayment for these surface helices is certain to increase the dynamic disorder of the LBD, as will be revealed in the next section.



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Figure 3. Comparison of the Arg 316 His Hormone-Binding Pocket with the Native Structure

Structural elements of the polar region of the hormone-binding pocket are displayed in stick representation. Carbon atoms are green for the receptor and gray for the hormone. Nitrogen, oxygen, and iodine are blue, red, and purple respectively. A, The buried polar cluster of the native hTRß. Arg 316 forms three side chain hydrogen bonds. One is with Gln 374 (part of helix 9); the other two hydrogen bonds are with residue Thr 232 in helix 1. B, Substitution of His at position 316 preserves the hydrogen bond with Gln 374 but eliminates both of the hydrogen bonds with Thr 232.

 
Arg 316 His and Ala 317 Thr Have Disordered Structure in Localized Regions
Figure 4Go shows a schematic representation of the B values, which measure disorder, for the native and mutated receptors. Observed B values for the Arg 316 His mutant are 27 Å2 higher than for the native structure. Because of the medium resolution of this structure determination, the absolute values cannot be reported with certainty. However, disorder or flexibility is clear in helix 0, the loop after helix 0, the loop after helix 1, helix 2, and the loop after it, continuing into helix 3, and the ß-hairpin. The average B values for Ala 317 Thr are as observed in the native structure. For both of the mutant receptors, loop 1 (between helices 1 and 2) exhibited significantly higher relative B values. Additional loops, normally flexible in the native structure, such as the loop before helix 12, may be more disordered. Helix 12 shows little change in relative B value between mutant and native structure (see Fig. 4BGo).



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Figure 4. Comparison of Normalized Thermal B Values between Native, Ala 317 Thr, and Arg 316 His

The B value range is represented by both coil radius and color change. The mean value and below is colored green and is 1 Å in radius. The top of the range is colored red and is 3 Å in radius. A, Normalized B values of the native hTRß model. The regions of the receptor that display the highest B values are helix 0, the loop after helix 1 and continuing into helix 3, and the loop between helix 11 and helix 12. B, Normalized B values of the Arg 316 His mutant. The regions of the receptor that display the highest B values are helix 0, the loop after helix 1 and continuing into helix 3, the ß-hairpin, and the loop linking helix 11 to helix 12. C, Normalized B values of the Arg 316 His mutant. The regions of the receptor that display the highest B values are helix 0, the loop after helix 0 and continuing into helix 3, and the loop between helix 11 and helix 12.

 
Ala 317 Thr and Arg 316 His Have Impaired Hormone Binding
Hormone binding assays for the native receptor, Ala 317 Thr, and Arg 316 His were performed using the LBD construct of hTRß with T3 to determine the hormone on-rate (kon), hormone off-rate (koff), and apparent equilibrium dissociation constant (Kd). Both Ala 317 Thr and Arg 316 His LBDs bound T3 with a lower affinity than the native receptor as reflected by almost 100-fold higher Kd values at 4° [5.7 (±5.0) x 10-11 for the native TR, vs. 4.7 (±0.9) x 10-9 and 2.0 (±0.3) x 10-9 for Ala 317 Thr and Arg 316 His, respectively; Table 2Go]. For the native TR and Arg 316 His, the apparent equilibrium Kd values are in agreement with previous studies (12, 18). The data in Table 2Go show that the binding ratio of T3 for Ala 317 Thr [Kd = 4.7 (±0.9) x 10-9] relative to the native receptor [Kd =5.7 (±5.0) x 10-11] of 0.008 is somewhat lower than that of 0.025 reported by Cheng et al. (19) and substantially lower than that of Hayashi et al. (20). These assays also showed that koff changed, whereas kon remained the same for both the native and mutant TRs. In agreement with the structural models, the binding data suggest that the mutant LBD-hormone complexes are less stable than the normal LBD-hormone complex, permitting faster dissociation of the hormone for both mutants.


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Table 2. Receptor Hormone Equilibrium Dissociation Constants and Association, Dissociation Rate Constants and Functional Characteristics

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
X-ray crystal structures of two mutated TR receptor LBDs demonstrate distinct mechanisms whereby the mutations can impair both hormone binding, and other functions of the receptors. The mutations are found next to one another in the TR primary sequence; both mutations cause RTH, result in impaired hormone binding, and are associated with elevated levels of T4. However, the mutants differ in that the Arg 316 His mutant is defective in homodimer formation, and this defect cannot be overcome with addition of more hormone (17).

Mutation in the T3 Binding Pocket Alters the LBD Surface
The major structural change detected in Ala 317 Thr is in the hormone binding pocket. In the native structure, the side chain of Ala 317 forms part of a hydrophobic pouch, which accepts the 3-iodine of the thyroid hormone-proximal phenolic ring. The side chain of the threonine in the mutant receptor pushes against the proximal ring of the hormone, moving it to deform the T3 binding pocket by about 1 Å on the side opposite to the mutation. The receptor adapts by displacing the front face of the receptor, loop 1, helix 2 (~1 Å), loop 2 (~1 Å), and the ß-hairpin (~1 Å). Thus, the positional variation is evident primarily on the front face of the receptor. Flexibility is suggested by the observation that the B factors, an index of disorder or multiply occupied positions, are increased in the liganded Ala 317 Thr LBD in the regions on either side of helix 1 [helix 0 (the C-terminal portion of the DBD), the loop after helix 1, helix 2 and the loop after it]. Thus, whereas crowding of the hormone in the T3 binding pocket of the receptor could explain the decreased affinity of the mutated receptor for T3, the instability of the receptor induced by the crowding may also contribute.

The perceived changes in flexibility have little functional consequence aside from T3 affinity. In studies in cell culture, increased hormone overcomes the defect and allows the mutated receptors to function as well, or almost as well, as the native receptors.

Arg 316 His Destabilizes Helix 1 and Other Receptor Structural Elements
The major influence of the Arg 316 His mutation is that the His 316 is unable to form hydrogen bonds made by the native Arg. In the native liganded receptor, Arg 316 is buried and inaccessible to solvent at the polar end of the hormone-binding pocket nearly 6 Å away from the carboxylate of the hormone. The Arg 316 side chain forms hydrogen bonds with helix 1 through the side chain and backbone of Thr 234, anchoring helix 1 to the body of the receptor and interlinking helices 1, 6, and 9. These changes result in a structure of the Arg 316 His mutant that differs more than the Ala 317 Thr does from native TR, primarily in the degree of disorder. The largest positional rearrangement is the section of helix 1 that normally would interact with Arg 316 in TR. A short section of helix 2, helix 3, and helix 0, leading into helix 1, accompanies the concerted shift in helix 1 away from His 316. Thus, as is the case for Ala 317 Thr, positional variation is evident primarily on the face of the receptor in the region of helices 1 and 2, further supporting the assertion of helix 1 being flexible. Helices 0 and 1 could also well be the critical DBD-LBD link that strengthens on hormone binding to the LBD. The instability induced by the His replacement of Arg is also evident from an analysis of the B factors, which are increased in Arg 316 His on either side of helix 1 and to a lesser degree in the loop between helix 11 and helix 12. These structural changes demonstrate that the position and stability of helix 1 is critical and suggest that the interactions of helix with the body of the receptor is important for receptor integrity, at least for binding affinity and probably for the associations between the DBD and LBD.

In this context, it is noteworthy that the Arg 316 His mutation results in impaired homodimer formation on DNA. This suggests that the stability of the helix 1 interaction with DNA is critical for this configuration of nuclear receptor dimer, although the precise requirement for helix 1 in this role is not clear. Dimer assembly without retinoid X receptor is mostly critical for unliganded TR, because on the direct repeat and inverted palindrome elements, there is little evidence of TR-TR homodimer formation on DNA even with wild-type receptors. In spite of the abnormalities detected in cells in culture, other functional defects, analogous to that with Ala 317 Thr, can be overcome with increased hormone, implying that the major effect of the mutation on the receptor is decreased affinity. In the cell, these instabilities otherwise do not prevent the receptor from working.

The existence of buried polar or hydrophilic networks of side chains is known in proteins and in protein interfaces. Their likely role is registration of structural elements and possible stabilization of the structure (21). Mostly, however, the free energy of stabilization of the protein structure derives from hydrophobic clusters buried from solvent. Nature may select the hydrophilic cluster in the TR LBD over a hydrophobic cluster to provide registration, stability, and the possibility of altering the interface in some functions of the receptor. Because the disruption of the buried hydrophilic cluster in TR causes a disease and affects the function dramatically, we compared other nuclear receptors. Surprisingly, we found that a buried cluster of polar side chains linking helix 1 and helix 9 (Table 3Go) is common to all nuclear receptors of known structure, although the identity of the pair varies between receptors. Such biological conservation of the biophysical polar feature where a hydrophobic feature is expected points to a critical role or functional advantage. The structures of the nuclear receptors do not reveal this role, which most likely is critical to flexibility or structural changes needed for exchange of binding partners.


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Table 3. Buried Polar Clusters in Nuclear Receptors

 
Helix 1 Interaction with Receptor Body Is Flexible
These data with both mutations emphasize the importance of the interaction between helix 1 and the body of the receptor. Previous work with LBD segments demonstrated that binding of helix 1 to the body of the receptor LBD is strengthened by hormone and corepressor (7). This is also consistent with the data from mutants with defective corepressor binding, Arg 243 Gln and Ala 234 Thr (22), which are internal, but could be disruptive in the same region of the LBD. The current studies with Ala 317 Thr demonstrate that displacing the hormone also translates into a shift in helix 1. These two observations are consistent with helix 1 loosely associating with the unliganded receptor and then folding into place more tightly when hormone is present. Recent data from the NMR structure of peroxisomal proliferator-activated receptor-{gamma} (PPAR{gamma}) suggest that both the C-terminal portion of helix 1 and the loop region after helix 1 are mobile in solution (8). In fact, it appears that the PPAR{gamma} receptor without hormone is in a partial molten globule state, where most of the hormone-binding pocket is disordered. Addition of hormone to PPAR allows the receptor to complete folding with formation of its remaining helices.

The RTH Mutations Decrease Affinity and Increase Hormone Off-Rate
We determined the binding constants for the mutant TRs (Table 2Go). The findings with the native and the Arg 316 His receptors were in agreement with earlier reports (12). However, the measured binding of T3 for Ala 317 Thr is weaker. The reasons are unclear.

We also determined the rates of association and dissociation of T3 from native and mutated receptors and found that the defect in both cases was in the rate of dissociation of hormone from the receptors (koff). The hormone on-rate for both mutants was similar to that of the native receptor. These results imply that the steric clash between the hormone and the hormone-binding pocket for Ala 317 Thr and the destabilization induced by the lack of hydrogen bonding for Arg 316 His do not impair the hormone’s ability to fit into the T3 binding pocket. The increased hormone off-rates are correlated with the increased relative B values in certain regions for both receptors. Both Ala 317 Thr and Arg 316 His have increased relative B values in the loop after helix 1 continuing into helix 3 and possibly in the loop after helix 11. It is emphasized that when bound, the hormone is completely enveloped by the receptor; thus, for dissociation to occur, the receptor must open to permit exit of the hormone. The mutations must increase the ease for this opening to occur, albeit by different mechanisms. The steric clash in the Ala 317 Thr mutation with the pressure on the body of the receptor on the side of the T3 binding pocket opposite to the mutation may induce receptor destabilization to allow dissociation. By contrast, with Arg 316 His the dynamic disorder induced by the electrostatic disruption of the polar cluster and the alteration of hydrogen bonds with helix 1 probably has a serious effect on hormone dissociation. Clearly the buried charges within the hormone-binding pocket play a significant role in the stability of the liganded receptor and may also be important in the stability of the unliganded receptor. Triac has one less positive charge than T3, and Arg 316 His has a diminished positive electrostatic potential within the hormone-binding pocket. The greater potency of Triac relative to T3 in promoting crystallization of the R316 mutant suggests that the electrostatic change in the binding pocket matters.

Changes Relative to Other X-Ray Crystal Structures Determined
We determined the structures of RTH TRs mutated away from the binding pocket, Ala 234 Thr, and Arg 243 Gln (23). These mutants were reported to be impaired in corepressor release, but with small decreases in T3 affinity. These mutations also modulate the flexibility and position of the loop after helix 1. The Ala 234 Thr mutation causes the tail of helix 1 to move away from the site of mutation and the residues that follow to reorganize their intramolecular interactions, resulting in increased flexibility of the protein chain after the mutation. The Arg 243 Gln mutation disrupts a weak surface salt bridge, allowing the loop after helix 1 to rotate away from the body of the receptor, causing it to become more mobile. The increased flexibility observed in the crystal structures of these two mutant proteins suggests that the conformational changes required to release corepressor is dependent on these chain segments being ordered after hormone binding. Although these mutations were reported to be only modestly impaired in their affinities for T3, we found that the affinity was decreased in both cases by almost 10-fold. In light of the current studies, this is not surprising, given that the stability of the receptor in this region should be important.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vectors
Mutant hTRß LBD extending from residue E202 to the C-terminal residue D461 was expressed in Escherichia coli as a His-tagged fusion protein using the vector pET28a (Novagen, Madison, WI). The PstI-BamHI fragments of the pET28 TRß E202 WT vector (10) were replaced with the PstI-BamHI fragments from the mutated TRs that were derived from pCDNA vectors (gift of Dr. S. Refetoff).

Protein Expression
Mutant hTRß LBD was expressed in E. coli strain BL21DE3 pLysS (Novagen). After growth in 2x concentrated Lennox broth (Sigma, St. Louis, MO) at 22 C to an OD600 = 1.5, 0.5 mM isopropyl ß-D thiogalactoside (Sigma) was added and growth continued for 6 h. Cells were harvested by centrifugation, and pellets were frozen in liquid nitrogen and stored at -80 C.

Purification
Thawed pellets from 1 liter culture were lysed in 50 mM sodium-phosphate (pH 8.0), 300 mM NaCl, 10% glycerol, 0.1% monothioglycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Benzamidine HCl, with 0.2 mg/ml lysozyme (20 min, 0 C). Extracts were sonicated briefly to break DNA and centrifuged (Ti45, 36000 rpm, 1 h, 4 C). After loading lysate on Talon resin (CLONTECH Laboratories, Inc., Palo Alto, CA) equilibrating in sodium phosphate buffer, the protein was eluted with 0–300 mM imidazole gradient. Isolation of liganded [3,3',5-triiodo-L-acetic acid (Triac; Sigma)] receptor using TSK-phenyl HPLC (TosoHaas, Philadelphia, PA) was performed as described (24); yield was 3 mg/liter bacterial culture. For crystallization, hTR ß LBD was diluted into 20 mM HEPES, pH 8.0, 3 mM dithiothreitol (DTT) and concentrated to 11.5 mg/ml by ultrafiltration (UFV2BGC10, Millipore Corp., Bedford, MA). The N-terminal His-tag was not removed before crystallization.

T3 Binding Saturation Assays
The affinities of binding of [125I]T3 to the hTRß LBD (WT and mutants) were determined at 4 C using saturation binding assays as described (10).

T3 Binding Kinetic Assays
For the dissociation experiments, 1 nM of [125I]T3 was allowed to bind 10 fmol of each receptor, at 4 C, until they had reached equilibrium (12 h). Adding unlabeled hormone in a concentration 100 times higher (100 nM) blocked further binding of radiolabeled hormone to the receptors. The receptor-bound [125I]T3 was isolated by gravity flow through a 2 ml coarse Sephadex G-25 (Pharmacia Biotech, Piscataway, NJ) column and quantified using a {gamma}-counter (COBRA, Packard Instruments, Meriden, CT). Binding curves were fit by nonlinear regression, and the koff values were calculated using the one-phase exponential decay equation contained in the Prism 3.0 program. The association experiments were performed using 2 nM of [125I]T3 and 10 fmol of receptor, at 4 C. After the receptor was added to the ligand, specific binding was measured immediately and at various times thereafter. Sample (100 µl) was applied to a Sephadex G-25 column at each time, and the eluate containing the bound receptor was quantified using a {gamma}-counter. Binding curves were fit by nonlinear regression and values of kob were determined by fitting an exponential association equation to the data. The association rate constant (kon), expressed in units of M-1 min-1, is expressed by kon = (kob - koff)/[radioligand]. The kon obtained experimentally was compared with the expected kon, calculated according to the law of mass action equation: Kd = koff/kon.

Crystallization and Preparation for Analysis
HTRß Ala317Thr.
Initial crystals of the Ala 317 Thr mutant bound to Triac were found using the previously established conditions for the TRß E202 histidine-tagged construct (10). Optimization of crystallization conditions resulted in a well buffer of 700 mM sodium acetate (NaH3OAc), 200 mM sodium succinate (NaSuc), and 100 mM sodium cacodylate (NaCac) adjusted to pH 7.2. Crystals were flash frozen in liquid nitrogen after the glycerol concentration was gradually increased through a series of soaks. The cryo solvents contained 900 mM NaH3OAc, 200 mM NaSuc, 100 mM NaCac, and a range of glycerol concentrations from 5–25% in five equal steps. These crystals diffracted to 2.4 Å at the Stanford Synchrotron Radiation Laboratory 7–1 beam line with a Mar 345 detector.

HTRß Arg 316 His.
The Arg 316 His mutant crystallized poorly in the same conditions found for native hTRß E202. Refinement of crystallization conditions produced optimal crystals at 800 mM NaH3OAc and 100 mM NaCac adjusted to pH 7.6. The combination of microseeding with TRß/T3 crystals, fresh unfrozen protein, and optimal growth conditions yielded one crystal that diffracted to 3.1 Å. Cryosolvent was the same as the mother liquor with 200 mM NaH3OAc added and a range of glycerol from 5–25% in five equal steps. One Arg 316 His crystal diffracted to 3.1 Å at the advanced light source beamline 5.0.2 with a detector from Area Detector Systems Corp. (Poway, CA).

Structural Refinement
HTRß Ala317Thr.
A data set for Ala 317 Thr was measured using one-degree oscillations through 120 degrees. The data set was found to be 93.3% complete to 2.4 Å resolution. Crystals of Ala 317 Thr/Triac exhibited the same hexagonal bipyramidal morphology found in wild-type receptor. Crystals displayed space group P3121 (a = 68.954 Å; c = 131.4 Å). Reflections were indexed and scaled using DENZO (HKL Research, Inc., Charlottesville, VA) and SCALEPACK (HKL Research, Inc.; Ref. 25). A molecular replacement solution was found with a Crystallography and NMR System 1 (Ref. 26 ; CNS 1.0) rotational search using the wild-type TR-ß/Triac structure with hormone and mutation region omitted. The structure was refined with multiple rounds of simulated annealing using CNS 1.0 and manual rebuilding with the Quanta98 software package. Electron density maps and coordinates were managed with the Collaborative Crystallography Project Number 4 in Protein crystallography package (27). Ten cycles of refinement were performed with REFMAC (Garib Murshudov, University of York) using a matrix weight of 0.1 resulting in a final model with an R factor of 22.2% and an Rfree of 25.9%.

HTRß Arg 316 His.
A data set for Arg 316 His was measured using one-degree oscillations through 180 degrees. This data set exhibited the same space group and morphology as the native data set. The crystal constants were slightly different (a = 67.274 Å; c = 130.121 Å). The data set for this mutant was reduced with DENZO. A simple rotational search with CNS 1.0 failed to produce an adequate molecular replacement solution. An independent molecular replacement search was performed with EPMR (Agouron Pharmaceuticals, Inc., La Jolla, CA; Ref. 28). The search converged on an acceptable solution after two cycles. The structure was refined using the same method described for Ala 317 Thr. However, the resulting model, with an R factor of 25.2% and an RFree of 30.2%, is less well defined. Many loop regions in the molecule are highly mobile and complicated the refinement. These regions could not be represented adequately using isotropic B values but showed sufficient electron density to prevent removal from the final model. We are confident that the molecular replacement solution was the correct one and the refinement was limited by the mobility of the loops. The best crystals for either mutant had a smallest dimension of 200 µm. The refined coordinates for both structures are deposited at the Protein Data Bank (http://www.rcsb.org/pdb/).


    ACKNOWLEDGMENTS
 
We would like to thank Dr. S. Refetoff for providing the hTRß cDNA containing the Ala 317 Thr and Arg 316 His mutations.


    FOOTNOTES
 
This work was supported by NIH Grants DK-41842, DK-09516, and DK-53417 (to J.D.B. and R.J.F.).

1 J.D.B. 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. Back

Abbreviations: CNS 1.0, Crystallography and NMR System 1; DBD, DNA-binding domain; hTRß, human thyroid hormone receptor-ß; LBD, ligand binding domain; NMR, nuclear magnetic resonance; PPAR, peroxisomal proliferator-activated receptor; RMSD, root mean square deviation; RTH, resistance to thyroid hormone; Triac, 3,5,3'-triiodothyroacetic acid.

Received for publication March 12, 2002. Accepted for publication January 2, 2003.


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 MATERIALS AND METHODS
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