Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

Abstract

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 Å 3 , effectively doubling the size of the GR dexamethasone-binding pocket of 540 Å 3 and yet leaving the structure of the coactivator binding site intact. DAC occupies only ϳ50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.Glucocorticoid receptor (GR) is a steroid hormone-regulated transcription factor that belongs to the nuclear receptor superfamily (1, 39). Upon ligand binding, GR regulates expression of an array of genes involved in glucose and lipid metabolism, bone turnover, lung maturation, and homeostasis of the immune, cardiovascular, and central nervous systems. GR ligands, including dexamethasone (DEX), fluticasone propionate, and other steroid analogs, are among the most effective agents for treating asthma, arthritis, leukemia, and various autoimmune diseases because of their potent anti-inflammatory and immunosuppressive effects. However, therapeutic use of glucocorticoids also induces a number of side effects including diabetes, bone loss, hypertension, and obesity (24). Although the molecular basis for these undesired side effects remains to be fully characterized (26), development of a GR ligand that can dissociate the therapeutic effects from the undesired adverse effects has been the subject of intense pharmaceutical research (23,25).The transcriptional function of GR is primarily controlled by ligand binding to its C-terminal ligand binding domain (LBD). In the absence of ligand, GR is retained in the cytoplasm by an association between the receptor LBD and the HSP90 chaperone complex (22). Ligand binding induces conformational changes in the LBD that lead to translocation of the receptor into the nucleus, where GR binds to DNA and regulates transcription of nearby genes. In addition to ...

Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor

“…This model required 2 residues (Arg-611 and Gln-570) to move substantially to accommodate the arylpyrazole moiety, and we noticed that this movement creates a new channel in the receptor extending from the meta-position of the phenyl ring. This expansion of the LBD and formation of the ''meta'' channel was supported by an in-house low-resolution GR crystal structure for a cortivazol analogue and has recently been confirmed by Suino-Powell et al with a well-resolved crystal structure for deacetylcortivazol 8 (22). The predicted binding mode for the arylpyrazole ligands has recently been confirmed with a nonsteroidal GR crystal structure for compound 5 in which the same new channel is again observed (23).…”

Insights from the structure of estrogen receptor into the evolution of estrogens: Implications for endocrine disruption

“…The ligand binding pocket on the GR can accommodate steroid analogs that are substantially larger than cortisol. In particular, deacylcortivazol, which has a large substituent at the A ring and thus lacks a C3 ketone, has a 10-fold higher affinity for the GR than does dexamethasone [46]. The crystal structure of the GR-deacylcortivazol complex Lastly, conformational flexibility of the ligand-binding pocket in the ER and other nuclear receptors may have been important in the evolution of their response to oxysterols and vertebrate hormones from ancestral signals [3,18,20].…”

Drug Design Approaches to Manipulate the Agonist-Antagonist Equilibrium in Steroid Receptors

“…An important lesson in this regard has been our change in understanding the dynamic nature of the steroid-receptor binding pocket. We have seen examples of extensive induced fits for amongst others the glucocorticoid receptor which is able to bind ligands beyond the conventional confines of its binding pocket whilst remaining in an agonistic conformation (Biggadike et al, 2009;Madauss et al, 2008;Suino-Powell et al, 2008). The pocket, behind the crucial helix-3 and helix-5 binding residues, Gln570 and Arg611, is normally water filled.…”