scholarly journals Dynamic allosteric communication pathway directing differential activation of the glucocorticoid receptor

2020 ◽  
Vol 6 (29) ◽  
pp. eabb5277 ◽  
Author(s):  
C. Köhler ◽  
G. Carlström ◽  
A. Gunnarsson ◽  
U. Weininger ◽  
S. Tångefjord ◽  
...  
Keyword(s):  
Communication Network ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Binding Pocket ◽  
Activation Function ◽  
Allosteric Control ◽  
Allosteric Communication ◽  
Helix 12 ◽  
Transmission Pathways ◽  
Communication Pathway

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

scholarly journals A Ligand Binding Domain Mutation in the Mouse Glucocorticoid Receptor Functionally Links Chromatin Remodeling and Transcription Initiation

1999 ◽  
Vol 19 (12) ◽  
pp. 8146-8157 ◽  
Author(s):  
Lynn A. Sheldon ◽  
Catharine L. Smith ◽  
Jack E. Bodwell ◽  
Allan U. Munck ◽  
Gordon L. Hager
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Activation Function ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Activation Process ◽  
Activation Domain

ABSTRACT We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR. With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays. Together, these findings support a two-step model for the transition of a nucleosome-assembled, repressed promoter to its transcriptionally active, derepressed form. In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain. These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.

scholarly journals AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors.

1997 ◽  
Vol 17 (4) ◽  
pp. 1832-1839 ◽  
Author(s):  
P M Henttu ◽  
E Kalkhoven ◽  
M G Parker
Keyword(s):  
Estrogen Receptor ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Alpha Helix ◽  
Steroid Receptor ◽  
Activation Function ◽  
Binding Activity ◽  
Lysine Residue ◽  
Steroid Receptor Coactivator ◽  
Helix 12

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.

scholarly journals Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains

2016 ◽  
Vol 30 (2) ◽  
pp. 173-188 ◽  
Author(s):  
Darrell E. Hurt ◽  
Shigeru Suzuki ◽  
Takafumi Mayama ◽  
Evangelia Charmandari ◽  
Tomoshige Kino
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Gene Mutations ◽  
Binding Pocket ◽  
Activation Function ◽  
Binding Activity ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Structural Defects ◽  
Mutant Receptors

Abstract Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR.

scholarly journals Probing Dominant Negative Behavior of Glucocorticoid Receptor β through a Hybrid Structural and Biochemical Approach

2018 ◽  
Vol 38 (8) ◽  
pp. e00453-17 ◽  
Author(s):  
Jungki Min ◽  
Lalith Perera ◽  
Juno M. Krahn ◽  
Christine M. Jewell ◽  
Andrea F. Moon ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Pocket ◽  
Dominant Negative ◽  
Ru 486 ◽  
Dominant Negative Inhibitor ◽  
Helix 12 ◽  
Binding Energy Analysis ◽  
Biochemical Approach ◽  
Biochemical Analyses

ABSTRACT Glucocorticoid receptor β (GRβ) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRβ functions as a dominant negative inhibitor of GRα at a molecular level, we determined the crystal structure of the ligand binding domain of GRβ complexed with the antagonist RU-486. The structure reveals that GRβ binds RU-486 in the same ligand binding pocket as GRα, and the unique C-terminal amino acids of GRβ are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRβ do not contribute to RU-486 binding. Intriguingly, the GRβ/RU-486 complex binds corepressor peptide with affinity similar to that of a GRα/RU-486 complex, despite the lack of helix 12. Our biophysical and biochemical analyses reveal that in the presence of RU-486, GRβ is found in a conformation that favors corepressor binding, potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRβ could repress transcription.

scholarly journals The Distinct Agonistic Properties of the Phenylpyrazolosteroid Cortivazol Reveal Interdomain Communication within the Glucocorticoid Receptor

2005 ◽  
Vol 19 (5) ◽  
pp. 1110-1124 ◽  
Author(s):  
Noritada Yoshikawa ◽  
Keiko Yamamoto ◽  
Noriaki Shimizu ◽  
Sachiko Yamada ◽  
Chikao Morimoto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Activation Function ◽  
Receptor Activation ◽  
Binding Domain ◽  
Fine Tuning ◽  
Docking Analysis ◽  
A Charge

Abstract Recent structural analyses of the nuclear receptors establish a paradigm of receptor activation, in which agonist binding induces the ligand binding domain (LBD)/activation function-2 helix to form a charge clamp for coactivator recruitment. However, these analyses have not sufficiently addressed the mechanisms for differential actions of various synthetic steroids in terms of fine tuning of multiple functions of whole receptor molecules. In the present study, we used the glucocorticoid receptor (GR)-specific agonist cortivazol (CVZ) to probe the plasticity and functional modularity of the GR. Structural docking analysis revealed that although CVZ is more bulky than other agonists, it can be accommodated in the ligand binding pocket of the GR by reorientation of several amino acid side chains but without major alterations in the active conformation of the LBD. In this induced fit model, the phenylpyrazole A-ring of CVZ establishes additional contacts with helices 3 and 5 of the LBD that may contribute to a more stable LBD configuration. Structural and functional analysis revealed that CVZ is able to compensate for the deleterious effects of a C-terminal deletion of the LBD in a manner that mimics the stabilizing influence of the F602S point mutation. CVZ-mediated productive recruitment of transcriptional intermediary factor 2 to the C-terminally deleted LBD requires the receptor’s own DNA binding domain and is positively influenced by the N-terminal regions of GR or progesterone receptor. These results support a model where ligand-dependent conformational changes in the LBD play a role in GR-mediated gene regulation via modular interaction with the DBD and activation function-1.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

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