Molecular dynamics simulations reveal multiple pathways of ligand dissociation from thyroid hormone receptors

Nuclear receptor (NR) ligands occupy a pocket that lies within the core of the NR ligand-binding domain (LBD), and most NR LBDs lack obvious entry/exit routes upon the protein surface. Thus, significant NR conformational rearrangements must accompany ligand binding and release. The precise nature of these processes, however, remains poorly understood. Here, we utilize locally enhanced sampling (LES) molecular dynamics computer simulations to predict molecular motions of x-ray structures of thyroid hormone receptor (TR) LBDs and determine events that permit ligand escape. We find that the natural ligand 3,5,3'-triiodo-L-thyronine (T(3)) dissociates from the TRalpha1 LBD along three competing pathways generated through i), opening of helix (H) 12; ii), separation of H8 and H11 and the Omega-loop between H2 and H3; and iii), opening of H2 and H3, and the intervening beta-strand. Similar pathways are involved in dissociation of T(3) and the TRbeta-selective ligand GC24 from TRbeta; the TR agonist IH5 from the alpha- and beta-TR forms; and Triac from two natural human TRbeta mutants, A317T and A234T, but are detected with different frequencies in simulations performed with the different structures. Path I was previously suggested to represent a major pathway for NR ligand dissociation. We propose here that Paths II and III are also likely ligand escape routes for TRs and other NRs. We also propose that different escape paths are preferred in different situations, implying that it will be possible to design NR ligands that only associate stably with their cognate receptors in specific cellular contexts.     

Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function

The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.     

Hormone selectivity in thyroid hormone receptors

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (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) TRalpha ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTRalpha LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRbeta receptor LBD in a complex with either Triac or a TRbeta-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTRalpha and hTRbeta LBDs show close structural similarity. However, the hTRbeta 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 (TRbeta) for Ser (TRalpha). 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 TRbeta 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 beta-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.     

Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.     

Molecular basis for dimer formation of TRbeta variant D355R

Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.     

Three-dimensional model of the ligand binding domain of the nuclear receptor for 1alpha,25-dihydroxy-vitamin D(3).

A three-dimensional model for residues 142-427 of the ligand binding domain (LBD) of the human nuclear receptor for 1alpha, 25-dihydroxy-vitamin D(3) [VDR] has been generated based on the X-ray crystallographic atomic coordinates of the LBD of the rat alpha1 thyroid receptor (TR). The VDR LBD model is an elongated globular shape comprised of an antiparallel alpha-helical triple sandwich topology, made up of 12 alpha-helical elements linked by short loop structures; collectively these structural features are similar to the characteristic secondary and tertiary structures for six nuclear receptors with known X-ray structures. The model has been used to describe the interaction of the conformationally flexible natural hormone, 1alpha,25-dihydroxy-vitamin D(3) [1alpha, 25(OH)(2)D(3)], and a number of related analogs with the VDR LBD. The optimal orientation of the 1alpha,25(OH)(2)D(3) in the LBD is with its A-ring directed towards the interior and its flexible side chain pointing towards and interacting with helix-12, site of the activation function-2 domain (AF-2) of the VDR. Mapping of four natural and one experimental point mutations of the VDR LBD, which result in ligand-related receptor dysfunction, indicates the close proximity of these amino acids to the bound ligand.