Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor

Abstract

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR)^1-3. Previously, we revealed that GR ligand binding activity is inhibited by Hsp70 and restored by Hsp90, aided by co-chaperones⁴. We then presented cryo-EM structures mechanistically detailing how Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding^5,6. In vivo, GR-chaperone complexes are found associated with numerous Hsp90 co-chaperones, but the most enigmatic have been the immunophilins FKBP51 and FKBP52, which further regulate the activity of GR and other steroid receptors^7-9. A molecular understanding of how FKBP51 and FKBP52 integrate with the GR chaperone cycle to differentially regulate GR activation in vivo is lacking due to difficulties reconstituting these interactions. Here, we present a 3.01 Å cryo-EM structure of the GR:Hsp90:FKBP52 complex, revealing , for the first time, that FKBP52 directly binds to the folded, ligand-bound GR using three novel interfaces, each of which we demonstrate are critical for FKBP52-dependent potentiation of GR activity in vivo. In addition, we present a 3.23 Å cryo-EM structure of the GR:Hsp90:FKBP51 complex, which, surprisingly, largely mimics the GR:Hsp90:FKBP52 structure. In both structures, FKBP51 and FKBP52 directly engage the folded GR and unexpectedly facilitate release of p23 through an allosteric mechanism. We also reveal that FKBP52, but not FKBP51, potentiates GR ligand binding in vitro, in a manner dependent on FKBP52-specific interactions. Altogether, we reveal how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation and how FKBP51 and FKBP52 compete for GR:Hsp90 binding, leading to functional antagonism.

Figure 1 |. Architecture of the GR:Hsp90:FKBP52 complex

a, Composite cryo-EM map of the GR:Hsp90:FKBP52 complex. Hsp90A (dark blue), Hsp90B (light blue), GR (yellow), FKBP52 (teal). Color scheme is maintained throughout. b, Atomic model in cartoon representation with boxes corresponding to the interfaces shown in detail in b-g. c, Interface 1 of the Hsp90:GR interaction, depicting the Hsp90A Src loop (Hsp90A^345-360) interacting with the GR hydrophobic patch. GR is in surface representation. d, Interface 2 of the Hsp90:GR interaction, depicting GR_{Helix 1} (GR^532-539) packing against the entrance to the Hsp90 lumen. Hsp90A/B are in surface representation. e, Interface 3 of the Hsp90:GR interaction, depicting GR_{pre-Helix 1} (GR^519-531) threading through the Hsp90 lumen. Hsp90A/B are in surface representation. f, Interface 1 of the Hsp90:FKBP52 interaction, depicting FKBP52 TPR H7e (FKBP52^387-424) interacting with the Hsp90A/B CTD dimer interface. Hsp90A/B are in surface representation. g, Interface 2 of the Hsp90:FKBP52 interaction, depicting the Hsp90B MEEVD motif (Hsp90B^700-706) binding in the helical bundle of the FKBP52 TPR domain. FKBP52 is in surface representation.

Figure 2 |. The GR:FKBP52 interaction and functional significance

a, Atomic model depicting the three interfaces between GR (yellow) and FKBP52 (teal) in the GR:Hsp90:FKBP52 complex. The FKBP52 proline-rich loop and PPIase site catalytic site are highlighted in gray. b, Interface 1 between GR (yellow) and the FKBP52 FK1 domain (teal), showing interacting side chains and hydrogen bonds (dashed pink lines). c, Interface 2 between GR (yellow) and the FKBP52 FK2 domain (teal), showing interacting side chains and hydrogen bonds (dashed pink lines). d, Interface 3 between GR (yellow) and the FKBP52 FK2-TPR linker (teal), showing interacting side chains and hydrogen bonds (dashed pink lines). e, GR activation assay in wild-type yeast strain JJ762 expressing FKBP52 (“52”) or FKBP52 mutants. The fold increase in GR activities compared to the empty vector (e.v.) control are shown (mean±SD). n=3 biologically independent samples per condition. Significance was evaluated using a one-way ANOVA (F_(6,14) = 67.82; p < 0.0001) with post-hoc Dunnett’s multiple comparisons test (n.s. P ≥ 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). P-values: p(e.v. vs. 52) < 0.0001, p(52 vs. 52ΔFK1) < 0.0001, p(52 vs. 52 S118A) < 0.0001, p(52 vs. 52 Y161D) = 0.0001, p(52 vs. 52 W259D) = 0.0002. f, Sequence alignment of eukaryotic FKBP52 showing conserved residues involved in the GR:FKBP52 interaction (denoted by a black asterisk). The bottom aligned sequence is human FKBP51. The alignment is colored according to the ClustalW convention. g, GR protein sequence conservation mapped onto the GR atomic model from the GR:Hsp90:FKBP52 complex. Residue conservation is depicted from most variable (cyan) to most conserved residues (maroon).

Figure 3 |. FKBP52 competes with p23 to bind GR:Hsp90

a, Atomic model of the GR-maturation complex (top) and the GR:Hsp90:FKBP52 complex (bottom) with boxes corresponding to the interfaces shown in detail in b-d. FKBP52 competes off p23 and re-positions GR at an approximately 45° rotated position. Hsp90A (dark blue), Hsp90B (light blue), GR (yellow), p23 (green), FKBP52 (teal). b, Position of the Hsp90A Src loop in the GR-maturation complex (Hsp90A, cyan) versus the GR:Hsp90:FKBP52 complex (Hsp90A, dark blue). The Hsp90A Src loop flips up in the GR:Hsp90:FKBP52 complex to interact with the hydrophobic patch on the rotated GR (yellow, surface representation). Hsp90A Src loop residues interacting with the GR hydrophobic patch are shown. c, Interface between the p23 tail-helix (green) and the GR hydrophobic patch (yellow, surface representation) in the GR-maturation complex (top). The p23 tail-helix is replaced by the Hsp90A Src loop (dark blue) in the GR:Hsp90:FKBP52 complex (bottom), which flip up to interact with the GR hydrophobic patch (yellow, surface representation). Interacting side chains are shown. d, Interaction between the GR_{pre-Helix 1} (GR^523-531) threading through the Hsp90 lumen in the GR-maturation complex (top) versus the GR_{pre-Helix 1} (GR^519-531) threading through the Hsp90 lumen in the GR:Hsp90:FKBP52 complex (bottom). Hsp90A/B are in surface representation colored by hydrophobicity. The GR_{pre-Helix 1} region translocates through the Hsp90 lumen by 2 residues in the transition from the GR-maturation complex to the GR:Hsp90:FKBP52 complex. Two hydrophobic residues on GR_{pre-Helix 1} (GR^L525,L528 or GR^P522,P526) remain bound in the Hsp90 lumen hydrophobic pockets in both complexes. e, Equilibrium binding of 10nM fluorescent dexamethasone to 100nM GR DBD-LBD with chaperones and FKBP52 (“52”). “Chaperones”= 15uM Hsp70, Hsp90, Hop, and p23 or p23Δhelix, 2uM Ydj1 and Bag-1. Significance was evaluated using a one-way ANOVA (F_(3,8) = 541.2; p < 0.0001) with post-hoc Šídák’s test (n.s. P ≥ 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). P-values: p(Chaperones vs. Chaperones) + 52 = 0.0002, p(Chaperones + 52 vs. Chaperones w/ p23Δhelix + 52) < 0.0001, p(Chaperones w/ p23Δhelix vs. Chaperones w/p23Δhelix + 52) < 0.0001. f, Equilibrium binding of 10nM fluorescent dexamethasone to 100nM GR DBD-LBD with chaperones, FKBP52 (“52”), and sodium molybdate (“Mo”). “Chaperones” = 15uM Hsp70, Hsp90, Hop, and p23, 2uM Ydj1 and Bag-1. Significance was evaluated using a one-way ANOVA (F_(5,12) = 761.5; p < 0.0001) with post-hoc Šídák’s test (n.s. P ≥ 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). P-values < 0.0001 for each comparison.

Figure 4 |. Architecture of the GR:Hsp90:FKBP51 complex

a, Composite cryo-EM map of the GR:Hsp90:FKBP51 complex. Hsp90A (dark blue), Hsp90B (light blue), GR (yellow), FKBP51 (purple). Color scheme is maintained throughout. b, Atomic model in cartoon representation with boxes corresponding to the interfaces shown in detail in c-e. c, Interface 1 between GR (yellow) and the FKBP51 FK1 domain (purple), showing interacting side chains and hydrogen bonds (dashed pink lines). d, Interface 2 between GR (yellow) and the FKBP51 FK2 domain (purple), showing interacting side chains and hydrogen bonds (dashed pink lines). e, Interface 3 between GR (yellow) and the FKBP51 FK2-TPR linker (yellow), showing interacting side chains and hydrogen bonds (dashed pink lines). f, Equilibrium binding of 10nM fluorescent dexamethasone to 100nM GR DBD-LBD with chaperones, FKBP51 (“51”), FKBP52 (“52”), or mutants. “Chaperones”= 15uM Hsp70, Hsp90, Hop, and p23 or p23Δhelix, 2uM Ydj1 and Bag-1. Significance was evaluated using a one-way ANOVA (F_(5,12) = 404.1; p < 0.0001) with post-hoc Šídák’s test (n.s. P ≥ 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P < 0.0001). See Methods for p-values.

Figure 5 |. Mechanism of GR regulation by FKBP51 and FKBP52 in the GR chaperone cycle in vivo

Schematic of the GR chaperone cycle in the cell. Starting on the top left, GR (yellow, cartoon representation) is in dynamic equilibrium between cortisol-bound and unbound (apo) states. Hsp70 (orange) binds GR and locally unfolds GR to inhibit cortisol-binding, stabilizing GR in a partially unfolded, apo state. Hsp70 transfers the partially unfolded GR to Hsp90 (light and dark blue):Hop (pink) to form the GR-loading complex (Wang et al. 2022), in which GR is stabilized in a partially unfolded, apo state with the cortisol-binding pocket accessible. Cortisol (pink), which enters the cell through diffusion, binds to GR during the transition from the GR-loading complex to the GR-maturation complex when Hsp90 refolds the GR to a native conformation, sealing the cortisol-binding pocket through the refolding of the GR Helix 1 region (Noddings et al. 2022). In the GR-maturation complex, the cortisol-bound, folded GR is stabilized by Hsp90 and p23 (green), and is protected from Hsp70 rebinding. Depending on the relative concentrations of the FKBPs, either FKBP51 (purple) or FKBP52 (teal) can bind the GR:Hsp90:p23 complex, competing off p23, and stabilizing the rotated position of GR. FKBP51 sequesters GR:Hsp90 in the cytosol until ATP hydrolysis on Hsp90 allows release of GR back to the chaperone cycle. In contrast, FKBP52 promotes rapid nuclear translocation of GR:Hsp90 by acting as an adapter to the dynein/dynactin motor complex. Once in the nucleus, the cortisol-bound GR can dimerize, nucleate the assembly of transcriptional regulatory complexes, and activate the transcription of thousands of genes, including the gene for FKBP51 (FKBP5), leading to a negative feedback loop that regulates GR activity in the cell. The GR chaperone cycle also occurs in the absence of ligand and evidence supports preferential binding of FKBP51 over FKBP52 to apo GR:Hsp90 complexes, insuring the apo (inactivated) GR is not improperly translocated to the nucleus to regulate transcription.