Modulation of the Hsp90 Chaperone Cycle by a Stringent Client Protein Oliver Robin Lorenz, Lee Freiburger, Daniel Andreas Rutz, Maike Krause, Bettina Karolina Zierer, Sara Alvira, Jorge Cuéllar, José María Valpuesta, Tobias Madl, Michael Sattler, Johannes Buchner  Molecular Cell  Volume 53, Issue 6, Pages 941-953 (March 2014) DOI: 10.1016/j.molcel.2014.02.003 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of the Purified GR-LBD (A) Domain architecture of the human GR. NTD indicates N-terminal domain; DBD indicates DNA-binding domain; hinge indicates hinge region; LBD indicates ligand binding domain. Domain borders as indicated. (B) Hsp90 dependence of GR mutants; shown are the averaged β-galactosidase activities and SDs of three independent experiments with each GR variant. Black bars show β-galactosidase activities of DMSO-treated control cells and red bars show remaining activity after treatment with 20 μM Radicicol. (C) Secondary structure of apo- and holo-GR-LBDm. (D) AUC sedimentation velocity analysis of apo- and holo-GR-LBDm. (E) Thermal stability of GR-LBDm was followed by CD spectroscopy. (F) Hormone binding affinity of GR-LBDm. Data was fit by a ligand-depletion model; inset shows binding kinetics of apo-GR-LBDm to F-DEX. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 GR-LBDm Binds Preferentially to Closed Hsp90 Conformations (A) Nucleotide-induced Hsp90 conformations affect GR-binding; AUC sedimentation velocity experiments were performed using ∗GR-LBDm; normalized dc/dt values were plotted against s20,W/S values. See also Figure S1A for the specificity of the interaction, Figure S1B for binding of GR-LBD F602S to Hsp90, and Figure S1C for conservation in the human system. (B) GR-LBDm binds with different affinity to nucleotide-induced conformations of Hsp90; shown are AUC sedimentation velocity titrations with ∗GR-LBDm using increasing amounts of Hsp90. The normalized concentration of bound GR-LBDm was plotted against the Hsp90 concentration. Data was fit according to a single-site binding model. (C) SAXS data showing a comparison of the experimental radial density distributions of Hsp90-ATP and with increasing stoichiometric ratios of GR-LBDm as indicated. (D) SAXS data showing a comparison of the experimental radial density distributions of Hsp90-GR-LBDm complexes in the absence and presence of nucleotides. For experimental radial density distributions of Hsp90 alone with different nucleotides, see Figure S1D. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 NMR Analysis of the Hsp90-GR Interaction (A) Zoomed views of 1H,15N HSQC experiments for Hsp90-N (top) or Hsp90-M (bottom) domains free (black) or with one molar equivalent of GR-LBDm in red. (B) Zoomed view of 1H,15N HSQC experiments for Hsp90-M in the presence of GR-LBDm spin labeled with IPSL oxidized (black) or reduced (red). (C) Zoomed view of 1H,15N HSQC experiments for Hsp90-C free (black) or bound to GR-LBDm (red). (D) CSPs and disappearing peaks observed in the titrations shown in (A) are highlighted as blue, green, and orange spheres for amides in the Hsp90-N, Hsp90-M, and Hsp90-C domains, respectively, and red spheres for peaks that experienced 20% bleaching (red) in the presence of oxidized spin label, mapped on a monomer in the structure of the Hsp90 closed dimer (PDB: 2CG9). (E) Zoomed views of 1H,15N correlations for segmentally isotope-labeled Hsp90-NM domain constructs, where either the -N (top) or -M (bottom) domain is detected free (black) or bound to GR-LBDm (red). (F) CSPs and disappearing peaks observed upon titration of the Hsp90-NM construct are indicated based on the NMR titrations shown in (E); color code as in (D). (G) Superposition of 1H,15N HSQC spectra of 15N-labeled GR-LBDm free (black) or bound to the Hsp90-M (green) and Hsp90-NM (red) domains. N- and NM-domain constructs were measured in the presence of AMP-PNP. For full-spectra, CSP intensity ratios, and spectra of isolated Hsp90-N, Hsp90-M, Hsp90-C, and Hsp90-NM constructs and GFP, see Figure S2 and Table S2. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 GR-LBDm Modulates the ATPase and the Conformational Cycle of Hsp90 (A) GR-LBDm decreases the ATPase activity of Hsp90 in a concentration-dependent manner; The mean values of three independent measurements were plotted; error bars as indicated. See Figure S3A for the effect of GR-LBD-F602S on the Hsp90 ATPase, Figure S3B for modulation of the ATPase activity of a disulfide-bridged Hsp90-NM construct by GR-LBDm, and Figure S3C for ATPase activity in the presence of the nonclient protein GFP. (B and C) GR-LBDm decreases the nucleotide-induced closing reaction of Hsp90 in a concentration-dependent manner; closing kinetics upon addition of ATPγS in the presence and in the absence of GR-LBDm. The relative Hsp90 closing rate was plotted against increasing amounts of GR-LBDm. Please see Figure S3D for the influence of GR-LBD-F602S on the nucleotide-induced closing reaction of Hsp90. (D) GR-LBDm stabilizes a partially closed conformation of Hsp90; FRET-chase kinetics upon the addition of unlabeled Hsp90 or ADP in the presence or absence of GR-LBDm. The reason for its biphasic nature is not clear, but it likely results from conformational rearrangements in Hsp90, as they are also observed in the absence of GR-LBDm. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Cochaperones Alter the GR-LBDm-Hsp90 Interaction (A) Cochaperones influence the affinity of GR-LBDm for Hsp90. Shown are AUC sedimentation velocity experiments for different cochaperone combinations. The differences in the s values observed for identical complexes do not reflect differences in their composition. (B) Hsp90 ATPase activity in the presence of GR-LBDm and various cochaperones determined from three independent measurements. Error bars as indicated. (C) SAXS data showing a comparison of the experimental radial density distributions of Hsp90-ATPγS at increasing stoichiometric ratios of GR-LBDm and p23 as indicated. For complex formation of Hsp90 with p23, see Figure S4. (D) Excess ADP disrupts GR-LBDm-Hsp90 and cochaperone complexes. The fractions of complexed ∗GR-LBDm were calculated from AUC sedimentation velocity experiments and plotted for each combination. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Structural Models for Hsp90-GR-LBDm and Hsp90-p23-GR-LBDm Complexes (A) EM reconstruction of the Hsp90-GR-LBDm complex. See also Figure S5 for further information. (B and C) Representation of Hsp90-GR-LBDm and Hsp90-GR-LBDm-p23 complexes. For lowest energy structures from the rigid body modeling calculations of free Hsp90, Hsp90-GR-LBDm, Hsp90-p23-GR-LBDm, and comparison of experimental radial density distribution with theoretical radial density distributions, see Figure S6. (D and E) Surface representation of Hsp90 showing the overlap with reported mutations (Bohen and Yamamoto, 1993; Fang et al., 2006; Genest et al., 2013; Nathan and Lindquist, 1995) and comparison of binding sites for the cochaperones Aha1 and Sti1 with the GR-LBDm binding site (Retzlaff et al., 2010; Schmid et al., 2012). The GR-LBDm binding site is shown in orange, surface exposed mutations are shown in magenta. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Integration of the GR into the Hsp90-Cochaperone Cycle Two scenarios exist for feeding GR into the Hsp90 machinery. In the case of apo-GR, the client assembles first with Hsp70 and Hsp40. After transfer to the Hsp90 chaperone machinery via Hop/Sti1, GR binds hormone. In the next step, Hsp70 release, together with the entry of a PPIase such as Cpr6, accelerates the cycle, resulting in the formation of a partially closed asymmetric holo-GR-Hsp90-PPIase-Hop/Sti1 complex. Then Hop/Sti1 is released by the concerted action of Cpr6 and p23, forming the late complex consisting of Hsp90, PPIase (Cpr6), p23, and holo-GR. Binding of p23 induces further closure of the Hsp90 dimer and reduces the ATPase activity of Hsp90, prolonging the interaction between the client and its chaperone. In the second scenario, holo-GR is bound directly by Hsp90. Whether the conformation of GR bound directly or delivered via Hsp70 differs is an open question at the moment. In the absence of cochaperones, Hsp90 is able to accommodate two GR molecules via two independent binding sites. Binding of p23 or Cpr6 to the Hsp90-holo-GR complex changes GR binding stoichiometry. Hydrolysis of ATP leads to the release of cochaperones and client. Molecular Cell 2014 53, 941-953DOI: (10.1016/j.molcel.2014.02.003) Copyright © 2014 Elsevier Inc. Terms and Conditions