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Cited by 137Heat shock proteins researchReceptor Mechanisms and SignalingProtein Structure and Dynamics

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Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex

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Gruszczyk, Jakub, Grandvuillemin, Loïc, Lai-Kee-Him, Josephine, Paloni, Matteo, Savva, Christos G., Germain, Pierre, Grimaldi, Marina, Boulahtouf, Abdelhay, Kwong, Hok-Sau, Bous, Julien, Ancelin, Aurélie, Bechara, Cherine, Barducci, Alessandro, Balaguer, Patrick, Bourguet, William·2022·Nature Communications
DOI 10.1038/s41467-022-34773-wPMC PMC9668932

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Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites. However, the scarcity of structural data precludes understanding of how AHR is activated by such diverse compounds. Our 2.85 Å structure of the human indirubin-bound AHR complex with the chaperone Hsp90 and the co-chaperone XAP2, reported herein, reveals a closed conformation Hsp90 dimer with AHR threaded through its lumen and XAP2 serving as a brace. Importantly, we disclose the long-awaited structure of the AHR PAS-B domain revealing a unique organisation of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing structural details of the molecular initiating event leading to AHR activation, our study rationalises almost forty years of biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

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Fig. 1 Overall architecture of the agonist-bound cytosolic complex of AHR. a A composite map of the Hsp90-XAP2-AHR complex in two orthogonal views. Hsp90A (light blue), Hsp90B (dark blue), XAP2 (orange), AHR (green). The same colour scheme is used throughout the manuscript unless stated otherwise. b The atomic model of the complex in cartoon representation (the orientation of the molecule same as in a). Nucleotide molecule, molybdate ion and indirubin ligand are shown in sticks.
Fig. 1 Overall architecture of the agonist-bound cytosolic complex of AHR. a A composite map of the Hsp90-XAP2-AHR complex in two orthogonal views. Hsp90A (light blue), Hsp90B (dark blue), XAP2 (orange), AHR (green). The same colour scheme is used throughout the manuscript unless stated otherwise. b The atomic model of the complex in cartoon representation (the orientation of the molecule same as in a). Nucleotide molecule, molybdate ion and indirubin ligand are shown in sticks.CC BY · PubMed Central
Fig. 2 Structure of the AHR PAS-B domain. a Two opposing views of the PAS-B domain in cartoon representation coloured in rainbow from N- to C-terminus. The elements of the secondary structure are labelled. The indirubin molecule is shown as sticks and coloured in magenta. b Close-up view showing PAS-B domain and its interaction with Hsp90 and XAP2. The contact sites of the PAS-B domain with the other components of the complex are coloured in green.
Fig. 2 Structure of the AHR PAS-B domain. a Two opposing views of the PAS-B domain in cartoon representation coloured in rainbow from N- to C-terminus. The elements of the secondary structure are labelled. The indirubin molecule is shown as sticks and coloured in magenta. b Close-up view showing PAS-B domain and its interaction with Hsp90 and XAP2. The contact sites of the PAS-B domain with the other components of the complex are coloured in green.CC BY · PubMed Central
Fig. 3 Details of the Hsp90-XAP2-AHR complex organisation. a An overall view of the complex with indicated location of the interaction sites between the proteins. The dashed circles indicate the contacts described in the following panels. b Two principal interaction sites between Hsp90 and PAS-B domains. c Close-up view of the AHR linker that is threaded through the Hsp90 lumen and interacts with both molecules. The residues for which we observed the strongest effect of the mutation on the complex stability in Co-IP assay are shown in small circles. d Results of Co-IP for the mutation of the AHR linker. The data were representative of three independent experiments. e Close-up view of the interaction sites between AHR and XAP2. f Co-IP results for the AHR mutants at the interface with XAP2. The data were representative of four independent experiments. g Interactions between XAP2 and Hsp90. h Co-IP results for the XAP2 mutants at the interface with Hsp90. The data were representative of two independent experiments. Source data are provided as a Source Data file.
Fig. 3 Details of the Hsp90-XAP2-AHR complex organisation. a An overall view of the complex with indicated location of the interaction sites between the proteins. The dashed circles indicate the contacts described in the following panels. b Two principal interaction sites between Hsp90 and PAS-B domains. c Close-up view of the AHR linker that is threaded through the Hsp90 lumen and interacts with both molecules. The residues for which we observed the strongest effect of the mutation on the complex stability in Co-IP assay are shown in small circles. d Results of Co-IP for the mutation of the AHR linker. The data were representative of three independent experiments. e Close-up view of the interaction sites between AHR and XAP2. f Co-IP results for the AHR mutants at the interface with XAP2. The data were representative of four independent experiments. g Interactions between XAP2 and Hsp90. h Co-IP results for the XAP2 mutants at the interface with Hsp90. The data were representative of two independent experiments. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Detailed analysis of the nucleotide binding site of Hsp90. a Close-up view of the nucleotide binding site in molecule A of Hsp90. The electron density map for the molecule of ADP, molybdate anion and magnesium cation has been displayed. b Close-up view of the molybdate ion. Increased contour level of the map density shows a much stronger density relative to the α and β-phosphates. c Details of the organisation of the nucleotide binding site. Amino acid residues involved in the interactions are shown as sticks and labelled. Hydrogen bonds are indicated as dashed green lines. d Schematic representation of the conformational cycle of Hsp90. Binding and release of a client protein is coupled with binding and hydrolysis of ATP. Molybdate mimics the presence of ATP and stabilises a closed conformation of Hsp90 even in the presence of ADP.
Fig. 4 Detailed analysis of the nucleotide binding site of Hsp90. a Close-up view of the nucleotide binding site in molecule A of Hsp90. The electron density map for the molecule of ADP, molybdate anion and magnesium cation has been displayed. b Close-up view of the molybdate ion. Increased contour level of the map density shows a much stronger density relative to the α and β-phosphates. c Details of the organisation of the nucleotide binding site. Amino acid residues involved in the interactions are shown as sticks and labelled. Hydrogen bonds are indicated as dashed green lines. d Schematic representation of the conformational cycle of Hsp90. Binding and release of a client protein is coupled with binding and hydrolysis of ATP. Molybdate mimics the presence of ATP and stabilises a closed conformation of Hsp90 even in the presence of ADP.CC BY · PubMed Central
Fig. 5 Detailed analysis of XAP2 protein and its interactions with the other partners. a Cartoon representation of XAP2 protein in two views coloured in rainbow from N- to C-terminus. Location of the most important features is indicated. XAP2 is composed of two domains that are separated by a short hinge. The N-terminal domain is not very well defined in the cryo-EM map most likely due to its increased mobility. The alpha-helical C-terminal domain is forming extensive contacts with both AHR and Hsp90 and is much better defined. PPI-like—peptidyl-prolyl isomerase-like domain. TPR1, 2 and 3—tetratricopeptide repeat motif 1, 2 and 3, α7—C-terminal helix alpha. b XAP2 is interacting extensively with two other proteins. Left panel: Imprint of the XAP2 on the other components of the complex. AHR and Hsp90 are shown in surface and XAP2 in cartoon representation; the AHR and Hsp90 residues in contact with XAP2 are coloured in orange. Right panel: Imprint of Hsp90 and AHR on XAP2. XAP2 is shown in surface, AHR and Hsp90 in cartoon representation. The XAP2 residues in contact with AHR and Hsp90 are coloured in green and blue, respectively. c Left panel: Imprint of the C-terminal part of Hsp90 on XAP2. Residues in contact with the MEEVD motif are coloured in dark blue. Right panel: Details of the XAP2 interactions with MEEVD motif of Hsp90. The C-terminal part of Hsp90 forms a helix that interacts with the TPR motifs. Hydrogen bonds are indicated as dashed blue lines.
Fig. 5 Detailed analysis of XAP2 protein and its interactions with the other partners. a Cartoon representation of XAP2 protein in two views coloured in rainbow from N- to C-terminus. Location of the most important features is indicated. XAP2 is composed of two domains that are separated by a short hinge. The N-terminal domain is not very well defined in the cryo-EM map most likely due to its increased mobility. The alpha-helical C-terminal domain is forming extensive contacts with both AHR and Hsp90 and is much better defined. PPI-like—peptidyl-prolyl isomerase-like domain. TPR1, 2 and 3—tetratricopeptide repeat motif 1, 2 and 3, α7—C-terminal helix alpha. b XAP2 is interacting extensively with two other proteins. Left panel: Imprint of the XAP2 on the other components of the complex. AHR and Hsp90 are shown in surface and XAP2 in cartoon representation; the AHR and Hsp90 residues in contact with XAP2 are coloured in orange. Right panel: Imprint of Hsp90 and AHR on XAP2. XAP2 is shown in surface, AHR and Hsp90 in cartoon representation. The XAP2 residues in contact with AHR and Hsp90 are coloured in green and blue, respectively. c Left panel: Imprint of the C-terminal part of Hsp90 on XAP2. Residues in contact with the MEEVD motif are coloured in dark blue. Right panel: Details of the XAP2 interactions with MEEVD motif of Hsp90. The C-terminal part of Hsp90 forms a helix that interacts with the TPR motifs. Hydrogen bonds are indicated as dashed blue lines.CC BY · PubMed Central
Fig. 6 Characterization of the interaction with indirubin. a Structure of indirubin. b Results of cell-based activity assays for four AHR ligands. The results are represented as mean value ± SD from independent biological replicates (n = 4). c Nano-DSF analysis of the interaction between Hsp90-XAP2-AHR (T, ternary) and Hsp90-XAP2-p23-AHR (Q, quaternary) complexes and indirubin. The results are representative of independent biological replicates (n = 3). d Two orthogonal views of the cryo-EM map for the region corresponding to indirubin. e Close-up view of the ligand binding pocket (LBP) shown as an orange surface. f Close-up view of the indirubin binding site. Residues interacting with the ligand are shown as magenta sticks. Hydrogen bonds are indicated as dashed blue lines. g Two layers of LBP residues determine AHR ligand selectivity. h Van der Waals (upper panel) and electrostatic (lower panel) interaction energies between indirubin and the residues of the protein. Data are presented as mean ± standard deviation of MD simulation replicas (n = 8). i Root mean square fluctuations (RMSF) of the protein backbone in bound (blue) and apo (red) conformations. Values are averages over the MD simulations; shaded areas represent the standard deviation between the replicas (n = 10 and n = 8 for the apo and bound system, respectively). j Two views of the PAS-B domain coloured according to the RMSF. The location of a potential ligand entry site to LBP is indicated with arrows. Source data are provided as a Source Data file.
Fig. 6 Characterization of the interaction with indirubin. a Structure of indirubin. b Results of cell-based activity assays for four AHR ligands. The results are represented as mean value ± SD from independent biological replicates (n = 4). c Nano-DSF analysis of the interaction between Hsp90-XAP2-AHR (T, ternary) and Hsp90-XAP2-p23-AHR (Q, quaternary) complexes and indirubin. The results are representative of independent biological replicates (n = 3). d Two orthogonal views of the cryo-EM map for the region corresponding to indirubin. e Close-up view of the ligand binding pocket (LBP) shown as an orange surface. f Close-up view of the indirubin binding site. Residues interacting with the ligand are shown as magenta sticks. Hydrogen bonds are indicated as dashed blue lines. g Two layers of LBP residues determine AHR ligand selectivity. h Van der Waals (upper panel) and electrostatic (lower panel) interaction energies between indirubin and the residues of the protein. Data are presented as mean ± standard deviation of MD simulation replicas (n = 8). i Root mean square fluctuations (RMSF) of the protein backbone in bound (blue) and apo (red) conformations. Values are averages over the MD simulations; shaded areas represent the standard deviation between the replicas (n = 10 and n = 8 for the apo and bound system, respectively). j Two views of the PAS-B domain coloured according to the RMSF. The location of a potential ligand entry site to LBP is indicated with arrows. Source data are provided as a Source Data file.CC BY · PubMed Central

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Authors
Gruszczyk, J., Grandvuillemin, L., Lai-Kee-Him, J., Paloni, M., Savva, C. G., Germain, P., Grimaldi, M., Boulahtouf, A., Kwong, H.-S., Bous, J., Ancelin, A., Bechara, C., Barducci, A., Balaguer, P., & Bourguet, W.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • University of Leicester (GB)
  • Inserm (FR)
  • Karolinska Institutet (SE)
Corresponding author
  • Gruszczyk, Jakub
  • Bourguet, William
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-11-16
Volume
13
Issue
1
Pages
7010-7010
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-022-34773-w
PMC
PMC9668932
OpenAlex
W4309473730
Cited by
137
References
58
Citations by year
2026: 38 · 2025: 28 · 2024: 39 · 2023: 31 · 2022: 1
FWCI
8.08
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • HAL (submittedVersion, cc-by-sa)
  • HAL (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • European Commission (825489, HORIZON2020)
  • Agence Nationale de la Recherche (ANR-16-IDEX-0006, ANR‐16‐IDEX‐, ANR-10-INBS-04-01, 10-INBS-04, 10-INBS-04-01, ANR‐10_INBS‐04, ANR-16, INBS-04-01, ANR-10, ANR-10-LABX-0012, ANR-10-INBS-0004)
  • Université de Montpellier (ANR_16-IDEX-0 0 06)
  • French Infrastructure for Integrated Structural Biology (ANR-10-INBS, ANR-10-INBS-04)
  • Région Occitanie Pyrénées-Méditerranée
  • Laboratoire d'Excellence EpiGenMed
  • Medical Research Council (MC_PC_17136, ANR-10-INBS-04)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
aryl hydrocarbon receptor, HSP90, indirubin, ligand binding, PAS-B domain, ligand specificity, receptor activation, transcription factors, structure-based drug design
MeSH terms
Cytosol, Humans, Ligands, Receptors, Aryl Hydrocarbon, HSP90 Heat-Shock Proteins, Cryoelectron Microscopy, Intracellular Signaling Peptides and Proteins, Basic Helix-Loop-Helix Proteins

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