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Large-scale, in-cell photocrosslinking at single-residue resolution reveals the molecular basis for glucocorticoid receptor regulation by immunophilins

Nature Structural & Molecular Biology volume 30, pages 1857–1866 (2023)Cite this article

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Abstract

The Hsp90 co-chaperones FKBP51 and FKBP52 play key roles in steroid-hormone-receptor regulation, stress-related disorders, and sexual embryonic development. As a prominent target, glucocorticoid receptor (GR) signaling is repressed by FKBP51 and potentiated by FKBP52, but the underlying molecular mechanisms remain poorly understood. Here we present the architecture and functional annotation of FKBP51-, FKBP52-, and p23-containing Hsp90–apo-GR pre-activation complexes, trapped by systematic incorporation of photoreactive amino acids inside human cells. The identified crosslinking sites clustered in characteristic patterns, depended on Hsp90, and were disrupted by GR activation. GR binding to the FKBPFK1, but not the FKBPFK2, domain was modulated by FKBP ligands, explaining the lack of GR derepression by certain classes of FKBP ligands. Our findings show how FKBPs differentially interact with apo-GR, help to explain the differentiated pharmacology of FKBP51 ligands, and provide a structural basis for the development of improved FKBP ligands.

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Fig. 1: Large-scale in-cell photocrosslinking reveals large and defined interaction interfaces of FKBPs with GR.
Fig. 2: Large-scale photocrosslinking of the full-length GR in mammalian cells confirms the interaction of the GRLBD and co-chaperones FKBP51, FKBP52, and p23.
Fig. 3: FKBP51, FKBP52, and p23 interact with the apo-GR in a Hsp90-dependent manner.
Fig. 4: Inhibition of FKBPs only partially disrupts FKBP–GR interactions.
Fig. 5: Co-chaperones preferentially engage apo-GR in pre-activation complexes in human cells.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request. The following protein structures were used in this paper, accessed via the PDB: 5OMP, 7L7I, 7KRJ, 7KW7, 3O5R, 6TXX, and 5OBK. Source data are provided with this paper.

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Acknowledgements

We thank M. Weissenborn, P. Püllmann, and C. Ulpinnis (University of Halle) for suggestions and training on the Golden Gate mutagenesis protocol, I. Coin (University of Leipzig) for plasmids and suggestions for pBpa incorporation in mammalian cells, and J. Kolos and T. Heymann for samples of 18(S)-Me and SAFit2, respectively. We are indebted to M. Wilfinger and J.-P. Kahl for cloning GR mutants and helping to establish the ELISA, and to F. Halloy for preliminary work on reporter gene assays. This work was supported by funding from the HMWK (LOEWE-Schwerpunkt TRABITA) and the BMBF (16GW0290K) to F.H.

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  1. These authors contributed equally: Asat Baischew, Sarah Engel.

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  1. Department of Chemistry, Technical University Darmstadt, Darmstadt, Germany

    Asat Baischew, Sarah Engel, Martha C. Taubert, Thomas M. Geiger & Felix Hausch

  2. Centre for Synthetic Biology, Technical University Darmstadt, Darmstadt, Germany

    Felix Hausch

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Contributions

A.B. and S.E. designed and executed all photocrosslinking experiments and subsequent analysis. T.M.G. performed and analyzed the reporter gene assays. M.C.T. performed and analyzed translocation and control experiments. F.H. conceived the project. All authors interpreted the results. A.B., S.E., and F.H. wrote the manuscript, which was approved by all authors.

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Correspondence to Felix Hausch.

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Nature Structural & Molecular Biology thanks Francis O’Reilly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 In-cell photocrosslinking confirms the interaction between TPR domain of FKBP51 and the C-terminal domain of Hsp90.

a, Western blots of exemplary FKBP51 pBpa mutants expressed and photocrosslinked in HEK293 cells co-expressing HA-tagged Hsp90. UV light-induced HA-reactive bands at a size of approx. 160 kDa are indicative of the mutated position being in proximity to Hsp90. b+c, Hsp90-photoreactive positions (highlighted in green) in the TPR domain of FKBP51 (pale pink) identified by Western blotting as in a were mapped on the structure of the Hsp90-FKBP51 complex (PDB: 7J7I, p23 omitted for clarity). Hsp90 dimers are shown in blue, the C-terminal MEEVD peptide of Hsp90 is depicted as blue spheres, and the unresolved linker between the MEEVD motif and one of the C-terminal domains of the Hsp90 dimers is indicated by a dotted blue line. c, Closeup view of crosslinks between positions K385, S392, A398, and M412 of FKBP51 with Hsp90. d+e, Close-up view of the predicted interaction of A398 and M412 of FKBP51 with Hsp90. points. All western blots shown are only performed once.

Source data

Extended Data Fig. 2 Sequence alignment of FKBP51 and FKBP52 showing FKBP→GR interaction.

Structural elements were marked on the sequence. Crosslinks to HA-GR are highlighted in green and no crosslinks are indicated in red.

Extended Data Fig. 3 Photocrosslinking of the full-length GR in mammalian cells confirms the interaction of the GR ligand binding domain and Hsp90 and the co-chaperones FKBP51 and p23.

a, Western blots of full length GR pBpa mutants expressed, photocrosslinked in HEK293 cells co-overexpressing HA-tagged FKBP51, and concentrated by FLAG-IP. UV light-induced HA-reactive bands at a size of approx. 180 kDa are indicative of the mutated position being in proximity to FKBP51. b, Tested position of GR→FKBP51 interaction are shown on GRLBD (PDB: 7KRJ), green indicates crosslink detected, red indicates no crosslink detected at this position. c, Structure of the GRLBD (523–777, PDB: 7krj) with α-helices shown in red and yellow and key loops highlighted in blue. d Overview of GR→FKBP51 and GR→FKBP52 Crosslinks e, GR→P23 crosslinks were highlighted on the GRLBD structure (523–777, PDB: 7krj), with indication of secondary structure. f, Western blots of full length GR pBpa mutants expressed, photocrosslinked in HEK293 cells without HA-FKBP51 overexpression, and concentrated by FLAG-IP. UV light-induced FLAG-reactive bands and FLAG-reactive bands at a size of 130 kDa are indicative of the mutated position being in proximity to p23. points. All western blots shown are only performed once.

Source data

Extended Data Fig. 4 GR agonist/antagonist disrupt FKBPs interaction with GR.

GR antagonist (Mifepristone, Mif) treatment for 1 h disrupts FKBP51→GR (a) and FKBP52→GR crosslinks (b). c, GR agonist (Dexamethasone, Dex), GR antagonist (Mifepristone, Mif) and Hsp90 inhibitor (Geldanamycin, GA) treatment for 1 h disrupts GR→FKBP51 crosslinks. points. All western blots shown are only performed once.

Source data

Extended Data Fig. 5 GR agonist disrupts FKBPs interaction with GR.

a, GR agonist (Dexamethasone, Dex) treatment for 1 h disrupts GRK743pBpa → FKBP51 and GRN768pBpa → FKBP51 crosslinks in a dose-dependent manner. Individual data points are shown. (n = 3 independent biological replicates). b, Amber suppression control of Extended Data Fig. 6a, Individual data points are shown. (n = 3 biological replicates). c + d, Treatment with Dexamethasone for 1 h, after transfection with double amount of GR plasmid compared to Fig. 3h. HA signal (c) and FLAG signal (d) measured from the same samples. (n = 3 biological replicates). e + f, Treatment with Dexamethasone for 10 min, after transfection with double amount of GR plasmid compared to Fig. 3h. HA signal (e) and FLAG signal (f) measured from the same samples. (n = 3 biological replicates). g, Time course experiment with varying Dex treatment times, 125 nM or 2 µM Dex was used h + j, cell fractionation study, stimulation with 1 µM Dex, except no treatment control (NT), with varying treatment times. Cells lysates were fractionated into; WC- whole cell lysate, C-cytosolic and N-nuclear fractions. i, Dex stimulation of GR results in loss of Hsp90 binding (sample concentrated by FLAG-IP). points. All western blots shown are only performed once.

Source data

Extended Data Fig. 6 Inhibition of FKBPs only partially disrupts FKBP-GR interactions.

a, Treatment with the FKBP inhibitor (18(S)-Me) for 1 h disrupts FKBP51→GR crosslinks in the FK1 (D68, E75) but not the FK2 domain (Q210, Y159). b, Treatment with the FKBP inhibitor (18(S)-Me) for 1 h disrupts FKBP52→GR crosslinks in the FK1 (D68, D75) but not the FK2 domain (R210, Y161). Western blots of FKBP51 (a) and FKBP52 (b) pBpa mutants expressed and photocrosslinked in HEK293 cells co-overexpressing HA-tagged GR. UV light-induced HA-reactive bands at a size of approx. 180 kDa are indicative of the mutated position being in proximity to GR. c, Treatment with the FKBP inhibitor (SAFit2) for 1 h disrupts FKBP52→GR crosslinks in the FK1 (D68, D75) but not the FK2 domain (R210, Y161). Western blots of exemplary FKBP52 pBpa mutants expressed and photocrosslinked in HEK293 cells co-overexpressing HA-tagged GR. UV light-induced HA-reactive bands at a size of approx. 180 kDa are indicative of the mutated position being in proximity to GR. d, SAFit2-sensitivity and resistance of exemplary GR pBpa mutants, detected by Western blots after expression and photocrosslinked in HEK293 cells with co-overexpressing HA-tagged FKBP51. points. All western blots shown are only performed once.

Source data

Extended Data Fig. 7 Reporter Gene Assays.

a, GR transactivation was measured by reporter gene assays in HEK293 cells transiently co-transfected with the dual reporters pGL4.36 (MMTV-luc2p), pGL4.74 (TK-hRluc) as well as expression plasmids for human GR and optionally FKBP51 and/or a 3-fold excess of FKBP52. Luminescence of the dual reporters luc2p and hRluc was measured 24 h after treatment with Dex and optionally 18(S)-Me (a). The FKBP ligand 18(S)-Me does not block FKBP51-induced GR suppression. Each bar (mean ± SD) represents data from biological replicates (n = 6, grey dots). b, FK506 (cyan, from PDB-ID: 3O5R), SAFit2 (green, from PBD-ID: 6TXX), and compound 18 (yellow, a close homolog of 18(S)-Me, from PDB-ID: 5OBK) are overlayed in the structure of FKBP51FK1 (PDB-ID: 5OBK), the close-up is rotated for a better view insight the binding pocket.

Source data

Extended Data Fig. 8 Differential GR interactions for the FK1 domains of FKBP51 and FKBP52.

a, Close-up view of FKBP51FK1→GR interactions, shown from three different angles. Crosslink (green) and inactive positions (red) were mapped on the structure of FKBP51FK1 (PDB: 3O5Q). b, Close-up view of FKBP52FK1→GR interactions, shown from three different angles. Crosslink (green) and inactive positions (red) GR were mapped on the structure of FKBP52FK1 (4LAV). c, Sequence alignment of the FK1 domains of FKBP51 and FKBP52, with GR-photoreactive positions highlighted in green and tested inactive positions in red. (·) different residue, (I) same residue, (!) crosslinking behavior different, (* or *) crosslinking behavior similar.

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Baischew, A., Engel, S., Taubert, M.C. et al. Large-scale, in-cell photocrosslinking at single-residue resolution reveals the molecular basis for glucocorticoid receptor regulation by immunophilins. Nat Struct Mol Biol 30, 1857–1866 (2023). https://doi.org/10.1038/s41594-023-01098-1

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