scholarly journals GR chaperone cycle mechanism revealed by cryo-EM: reactivation of GR by the GR:Hsp90:p23 client-maturation complex

2020 ◽  
Author(s):  
Chari M. Noddings ◽  
Ray Yu-Ruei Wang ◽  
David A. Agard
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Molecular Mechanism ◽  
Molecular Chaperone ◽  
Complex Structure ◽  
Binding Domain ◽  
Atomic Resolution ◽  
Native Conformation ◽  
Client Proteins ◽  
Resolution Structure

AbstractHsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of ‘client’ proteins1,2. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity3. Previously, we revealed GR ligand binding is inhibited by Hsp70 and restored by Hsp90, aided by the cochaperone p234. However, a molecular understanding of the chaperone-induced transformations that occur between the inactive Hsp70:Hsp90 ‘client-loading complex’ and an activated Hsp90:p23 ‘client-maturation complex’ is lacking for GR, or for any client. Here, we present a 2.56Å cryo-EM structure of the GR-maturation complex (GR:Hsp90:p23), revealing that the GR ligand binding domain is, surprisingly, restored to a folded, ligand-bound conformation, while simultaneously threaded through the Hsp90 lumen. Also, unexpectedly, p23 directly stabilizes native GR using a previously uncharacterized C-terminal helix, resulting in enhanced ligand-binding. This is the highest resolution Hsp90 structure to date and the first atomic resolution structure of a client bound to Hsp90 in a native conformation, sharply contrasting with the unfolded kinase:Hsp90 structure5. Thus, aided by direct cochaperone:client interactions, Hsp90 dictates client-specific folding outcomes. Together with the GR-loading complex structure (Wang et al. 2020), we present the molecular mechanism of chaperone-mediated GR remodeling, establishing the first complete chaperone cycle for any client.

scholarly journals GR chaperone cycle mechanism revealed by cryo-EM: the GR-maturation complex

2020 ◽  
Author(s):  
David Agard ◽  
Chari Noddings ◽  
Ray Wang
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Molecular Mechanism ◽  
Molecular Chaperone ◽  
Complex Structure ◽  
Binding Domain ◽  
Atomic Resolution ◽  
Native Conformation ◽  
Client Proteins ◽  
Resolution Structure

Abstract Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of ‘client’ proteins. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity. Previously, we revealed GR ligand binding is inhibited by Hsp70 and restored by Hsp90, aided by the cochaperone p23. However, a molecular understanding of the chaperone-induced transformations that occur between the inactive Hsp70:Hsp90 ‘client-loading complex’ and an activated Hsp90:p23 ‘client-maturation complex’ is lacking for GR, or for any client. Here, we present a 2.56Å cryo-EM structure of the GR-maturation complex (GR:Hsp90:p23), revealing that the GR ligand binding domain is, surprisingly, restored to a folded, ligand-bound conformation, while simultaneously threaded through the Hsp90 lumen. Also, unexpectedly, p23 directly stabilizes native GR using a previously uncharacterized C-terminal helix, resulting in enhanced ligand-binding. This is the highest resolution Hsp90 structure to date and the first atomic resolution structure of a client bound to Hsp90 in a native conformation, sharply contrasting with the unfolded kinase:Hsp90 structure. Thus, aided by direct cochaperone:client interactions, Hsp90 dictates client-specific folding outcomes. Together with the GR-loading complex structure (Wang et al. 2020), we present the molecular mechanism of chaperone-mediated GR remodeling, establishing the first complete chaperone cycle for any client.

scholarly journals Folding and stability of the ligand-binding domain of the glucocorticoid receptor

2002 ◽  
Vol 11 (8) ◽  
pp. 1926-1936 ◽  
Author(s):  
Stephen H. McLaughlin ◽  
Sophie E. Jackson
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain

scholarly journals Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity

2003 ◽  
Vol 23 (6) ◽  
pp. 1922-1934 ◽  
Author(s):  
Marcel J. M. Schaaf ◽  
John A. Cidlowski
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Conformational Change ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Dependent Manner ◽  
Dna Binding Domain ◽  
Ligand Affinity ◽  
High Affinity

ABSTRACT The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR α-isoform (hGRα) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRα ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRα antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRα mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRα deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRα differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.

scholarly journals Farnesylation of Ydj1 Is Required for In Vivo Interaction with Hsp90 Client Proteins

2008 ◽  
Vol 19 (12) ◽  
pp. 5249-5258 ◽  
Author(s):  
Gary A. Flom ◽  
Marta Lemieszek ◽  
Elizabeth A. Fortunato ◽  
Jill L. Johnson
Keyword(s):  
Molecular Chaperone ◽  
Steroid Receptors ◽  
Binding Domain ◽  
Zinc Binding ◽  
Physical Interaction ◽  
Client Proteins ◽  
Carboxy Terminal ◽  
Zinc Binding Domain ◽  
Substrate Binding Domain

Ydj1 of Saccharomyces cerevisiae is an abundant cytosolic Hsp40, or J-type, molecular chaperone. Ydj1 cooperates with Hsp70 of the Ssa family in the translocation of preproteins to the ER and mitochondria and in the maturation of Hsp90 client proteins. The substrate-binding domain of Ydj1 directly interacts with steroid receptors and is required for the activity of diverse Hsp90-dependent client proteins. However, the effect of Ydj1 alteration on client interaction was unknown. We analyzed the in vivo interaction of Ydj1 with the protein kinase Ste11 and the glucocorticoid receptor. Amino acid alterations in the proposed client-binding domain or zinc-binding domain had minor effects on the physical interaction of Ydj1 with both clients. However, alteration of the carboxy-terminal farnesylation signal disrupted the functional and physical interaction of Ydj1 and Hsp90 with both clients. Similar effects were observed upon deletion of RAM1, which encodes one of the subunits of yeast farnesyltransferase. Our results indicate that farnesylation is a major factor contributing to the specific requirement for Ydj1 in promoting proper regulation and activation of diverse Hsp90 clients.

Enhancing the Stability and Solubility of the Glucocorticoid Receptor Ligand-Binding Domain by High-Throughput Library Screening

2010 ◽  
Vol 403 (4) ◽  
pp. 562-577 ◽  
Author(s):  
Tobias Seitz ◽  
Ralf Thoma ◽  
Guillaume A. Schoch ◽  
Martine Stihle ◽  
Jörg Benz ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
High Throughput ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Library Screening ◽  
Receptor Ligand ◽  
The Stability
Sign in / Sign up

Export Citation Format

Share Document