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.
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ABSTRACT |
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INTRODUCTION |
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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 receptors 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 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. 1) (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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RESULTS |
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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. 2. 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 0.30.5 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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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. 3. 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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DISCUSSION |
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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 3) 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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The RTH Mutations Decrease Affinity and Increase Hormone Off-Rate
We determined the binding constants for the mutant TRs (Table 2). 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 hormones 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.
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MATERIALS AND METHODS |
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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 0300 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 -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
-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 525% in five equal steps. These crystals diffracted to 2.4 Å at the Stanford Synchrotron Radiation Laboratory 71 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 525% 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/).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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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REFERENCES |
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