Conformational changes of the glucocorticoid receptor ligand binding domain induced by ligand and cofactor binding, and the location of cofactor binding sites determined by hydrogen/deuterium exchange mass spectrometry

Abstract

HXMS (hydrogen/deuterium exchange mass spectrometry) of the glucocorticoid receptor ligandbinding domain (GR LBD) complexed with the agonist dexamethasone and the antagonist RU-486 is described. Variations in the rates of exchange were observed in regions consistent with the published crystal structures of GR LBD complexed with RU-486 when compared with the GR dexamethasone complex. We also report the HXMS results for agonist-bound GR LBD with the coactivator transcriptional intermediary factor 2 (TIF2) and anatagonist-bound GR LBD with nuclear receptor corepressor (NCoR). Alterations in exchange rates observed for agonist-bound GR LBD with TIF2 present were consistent with the published crystal structural contacts for the complex. Alterations in exchange rates observed for antagonist-bound GR LBD with NCoR were a subset of those observed with TIF2 binding, suggesting a common or overlapping binding site for coactivator and corepressor. Keywords: glucocorticoid receptor; H/D exchange; mass spectrometrySupplemental material: see www.proteinscience.orgThe glucocorticoid receptor (GR) is a member of the nuclear receptor family; its members include the mineralcorticoid (MR), progesterone (PR), estrogen (ER), and androgen (AR) receptors (Robinson-Rechavi et al. 2001). The glucocorticoid receptor has been shown to regulate glucose homeostasis, lipid metabolism, and inflammation (Reichardt et al. 2000), making it a therapeutically important drug target. Marketed steroids such as dexamethasone and prednisolone are effective at suppressing inflammation but also show negative side effects, including diabetes, hyperglycemia, and osteoporosis (Schacke et al. 2002). Pharmaceutical research continues to seek high affinity glucocorticoid receptor ligands that have potent anti-inflammatory activity with reduced side effects. Much work has been done to identify novel inhibitors that show such dissociative behavior (Coghlan et al. 2003;Schacke et al. 2004).GR is a transcription factor that is activated upon ligand binding. Upon glucocorticoid binding, the GR receptor translocates from the cytoplasm into the nucleus, where it serves either directly or indirectly as a transcriptional regulator. Transrepression of genes is understood to be Reprint requests to: Lee Frego, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, CT 06877, USA; e-mail: lfrego@ rdg.boehringer-ingelheim.com; fax: (203) 791-6196.Abbreviations: H/D, hydrogen/deuterium; HXMS, hydrogen/deuterium exchange mass spectrometry; LC/MS, liquid chromatography/mass spectrometry; GR, glucocorticoid receptor; GR LBD, glucocorticoid receptor ligand-binding domain; TIF2, transcriptional intermediary factor 2; SRC1, steroid receptor coactivator 1; NCoR, nuclear receptor corepressor; NF-kB, nuclear factor kB; TAT, tyrosine aminotransferase; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; DBD, DNA-binding domain; LBD, ligand-binding domain; NMR, nuclear magnetic resonance.Article and publication are at

Differential modulation of glucocorticoid and progesterone receptor transactivation

“…These inverted responses were then shown to depend upon the joint actions of the N-and C-terminal domains of each receptor (Song et al, 2001). These results are consistent with the demonstration that corepressors interact with N-terminal regions of both GRs and PRs Wang et al, 2007) in addition to the initially defined requirements of the C-terminal sequences of nuclear (Zamir et al, 1996) and steroid receptors (Cheng et al, 2002;Wang et al, 2004a;Frego and Davidson, 2006;Wu et al, 2006;Kroe et al, 2007;Wang et al, 2007).…”

Antiinflammatory glucocorticoid receptor ligand with reduced side effects exhibits an altered protein–protein interaction profile

“…This profile is similar, but not identical, to the profile of a known antagonist, RU486. Interactions with corepressors have been documented for GR previously in response to antagonists (32,33). These results suggest that LGD5552-bound GR can adopt a conformation that also accommodates larger corepressor peptides (34).…”

Hormone Binding and Co-regulator Binding to the Glucocorticoid Receptor are Allosterically Coupled

“…To facilitate this ligand binding study, unlabeled dex remaining from the protein preparation was removed from solution through dialysis or desalt- ing. Previous studies (58,60,64) have shown that recombinantly expressed GR LBD can readily exchange ligand under non-denaturing conditions, in sharp contrast with the androgen receptor, 4 suggesting ligand binding pocket accessibility may be a feature of GR. Fig.…”

“…Biochemical interrogation of GR LBD and its various interaction partners has long been hindered by difficulty in obtaining stable, functional receptors for in vitro studies. The majority of published work using recombinantly expressed GR LBD, including this study, has been aided by incorporation of solubility-enhancing mutations discovered by yeast screens of GR activity (58,59,64), 3 and GR containing the mutations F602S and C638D will be referred to henceforth as GR SD .…”