Differential effects of TR ligands on hormone dissociation rates: Evidence for multiple ligand entry/exit pathways

Abstract

Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself. This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here, we show that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation (T_1/2 < 2 h) of radiolabeled T₃ vs. T₃ (T_1/2 ≈ 5–7 h). We cannot discern relationships between this effect and ligand size, activity or affinity for TRβ. One ligand, GC-24, binds the TR LBC and (weakly) to the TRβ-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T₃ dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T₃ release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T₃ release and that this effect enhances hormone dissociation.

Introduction

Nuclear receptors (NRs) regulate gene expression in response to small signaling molecules [1]. The NR family includes receptors for thyroid hormone (TH) [2], steroid hormones, vitamins A and D, cholesterol and fatty acid derivatives, heme, glucose and other molecules. Since NRs play widespread roles in development and disease, they are important targets for pharmaceutical discovery. TH receptors (TRs) are the subject of efforts to develop selective agonists to ameliorate aspects of metabolic syndrome without harmful effects on heart and antagonists to treat hyperthyroidism and other conditions [3], [4]. Improved understanding of mechanisms of NR ligand association and dissociation will provide insights into receptor function and could suggest ways to stabilize or destabilize bound hormone, improve antagonism and facilitate development of drugs that interact tightly and selectively with cognate NRs.

NRs harbor a single ligand binding cavity (LBC) whose location, relationship to gene activation and organization have been extensively studied [1], [5], [6]. X-ray structures of NR LBDs with agonists reveal that the LBC is buried in the C-terminal ligand binding domain (LBD). Agonists promote packing of C-terminal helix (H) 12 against the LBD to complete a coactivator binding surface, activation function 2 (AF-2) [5], [7]. Close investigation of the LBCs of the two TRs (TRα and TRβ) revealed one subtype specific amino acid in the TR LBC involved in ligand contact (TRβN331/TRαS277) and it has been possible to exploit this difference to obtain TRβ selective ligands [3], [8]. X-ray structures also reveal that the buried pocket is flexible; the TR LBC can expand to accommodate a bulky 5′ iodine substituent in the parental form of TH, thyroxine (T₄), and a bulky 3′ phenyl group in the TRβ selective agonist, GC-24 [6], [9], [10].

In contrast, mechanisms of ligand binding and dissociation from the LBC are only partly understood [11], [12]. X-ray structures of NR LBDs reveal that H12 can move to expose the LBC, and this probably constitutes one ligand escape route [5], [7], [11], [12]. However, our analyses of regions of instability in X-ray structures [13], [14], [15] and molecular dynamics simulations [11], [12] suggest that active TH (triiodothyronine, T₃) can escape from the LBD in three ways: under H12 (Path I, described above); between H8 and H11 near the dimer/heterodimer surface at the H10/H11 junction (Path II); and through the H1–H3 loop (Path III). Our simulations also suggest that escape routes vary with ligand and receptor; T₃ prefers Path III whereas the TRβ-selective GC-24 prefers Path I [12].

While the notion that there are multiple ligand escape paths await definitive verification, a number of data are consistent with this conclusion. Structural elements that permit ligand escape through each pathway are implicated in stable agonist binding [11], [12]. For Path I, suboptimal packing of TRβ H12 against the T₄–LBD complex is associated with rapid ligand dissociation [9]. Conversely, point mutations and coactivators that stabilize estrogen receptor (ER) or TR H12 in the active position reduce hormone dissociation rates [16], [17], [18]. For Path II (involving residues near the dimer surface), resistance to thyroid hormone syndrome (RTH) mutations that affect this region enhance T₃ dissociation rates [19]. For Path III, X-ray structural analysis of other RTH mutants reveals that increased T₃ dissociation rate is associated with disorder in the H1–H3 loop [14], [15], [19].

In spite of strong evidence for a single high affinity hormone binding site, early studies raised the possibility that NRs harbor auxiliary ligand binding sites that exert allosteric effects on bound hormone. Some NR interacting compounds displace bound high affinity ligands more rapidly than the high affinity ligand itself. Progesterone, for example, binds glucocorticoid receptors (GRs) with lower affinity than dexamethasone, and acts as an antagonist, yet displaces this higher affinity agonist more rapidly than dexamethasone [20]. The mechanism of this effect is not clear, but it was proposed that progesterone binds to an undefined allosteric site to promote dexamethasone dissociation.

Recent evidence confirmed that there are multiple ligand binding sites on NR LBD surfaces. Several compounds bind to NR (including TR) AF-2 sites [21], [22], [23]. Other compounds were found at another location on the androgen receptor surface (BF-3) in X-ray screens and ligand binding to BF-3 may exert allosteric effects on AR AF-2 [24]. Finally, the TR agonist GC-24 binds to at a location near the TR dimer/heterodimer surface at the junction of TRβ H10 and H11 [10].

In this paper, we show that TR ligands (including GC-24) displace bound hormone at different rates and investigate this phenomenon. The effect is not related to ligand affinity, activity or size and does not appear to involve surface ligand interactions. Instead, several lines of evidence suggest that the challenger interacts with the LBC to displace bound hormone. We propose that challengers promote ligand release by binding partially unfolded conformational intermediates that occur in ligand release and blocking refolding of the hormone/receptor complex around labeled ligand. Implicit in this hypothesis is the concept that different ligands associate with TRs via different pathways.