ABSTRACT
Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5' extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5' iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRbeta structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3' benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.
Subject(s)
Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/chemistry , Acetates/chemistry , Acetates/metabolism , Amino Acid Sequence , Benzhydryl Compounds/chemistry , Benzhydryl Compounds/metabolism , Binding Sites/genetics , Conserved Sequence , Crystallography, X-Ray , Drug Design , Humans , In Vitro Techniques , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Thyroid Hormone/agonists , Receptors, Thyroid Hormone/chemistry , Receptors, Thyroid Hormone/genetics , Receptors, Thyroid Hormone/metabolism , Sequence Homology, Amino Acid , Thyroxine/chemistry , Thyroxine/metabolismABSTRACT
Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.
Subject(s)
Receptors, Thyroid Hormone/chemistry , Thyroxine/chemistry , Animals , CHO Cells , Chromatography , Cricetinae , Crystallography, X-Ray , DNA/chemistry , Dimerization , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Iodine/chemistry , Kinetics , Ligands , Models, Chemical , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Time Factors , Transfection , Triiodothyronine/chemistryABSTRACT
Resistance to hormones is commonly due to mutations in genes encoding receptors. Resistance to thyroid hormone is due mostly to mutations of the beta-form of the human (h) thyroid hormone receptor (hTRbeta). We determined x-ray crystal structures of two hTRbeta 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 hTRbeta. 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.
