Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques

Abstract

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

Keywords: DEER; EDNMR; ENDOR; Hsp90; chaperone.

Fig. 1.

Structure and conformational cycle of yHsp90 with the spin labels used in our study and their locations indicated. (A) X-ray structure of yHsp90/AMP-PNP/p23 [PDB ID code 2CG9 (32)], corresponding to the closed conformation, showing only the yHsp90 part with the N-, M-, C-domains indicated. The residues before mutation to cysteines and spin labeling are shown in sphere representation as follows: D61C in blue, A152C in pink, Q385C in red, E517C in yellow, D560C in cyan, and K637C in orange. In the N-domains the nucleotides are shown as sticks, while the metal cofactor shown as a light orange sphere was added to the structure as a pseudoatom coordinated by the N₇, β-, and γ-phosphates of AMP–PNP. (B) Postulated conformational cycle constructed from literature data (in the spirit of Taipale et al., figure 1 in ref. and Schulze et al., figure 6 in ref. 17). The arrows in the apo state denote a range of conformations the N-domains can adopt, while in the nucleotide-bound states Hsp90 is in an equilibrium between an open and a closed conformation. (C and D) Chemical structure of the spin labels used in the study after formation of a C-S (thio-sulfide) bond with a cysteine residue of the protein (in red).

Fig. 2.

W-band DEER distance measurements on the C-domain yHsp90 mutants (A) D560C/Gd(III) and (B) K637C/Gd(III). On the left are given the background-corrected DEER time traces with the fit shown in gray lines and on the right are the distance distributions with confidence intervals as analyzed in the DeerAnalysis software (63). Modeled distance distributions using the MtsslWizard software (42) and the closed X-ray structure [PDB ID code 2CG9 (32)] are shown in orange. For details of the data processing and modeling, see Methods. The apo-, pre-, and posthydrolysis states are in black, green, and blue, respectively. The background-colored areas in the distance distributions denote reliability regions as follows: green: shape reliable; yellow: mean and width reliable; orange: mean reliable; red: nonreliable. The data are shown with offset on the y axis. The DEER data were acquired with chirp pump pulses (–76, 78) (for set-up, see Methods and SI Appendix, Methods and Fig. S7C) and the primary DEER data are given in SI Appendix, Fig. S8.

Fig. 3.

The coordination environment of yHsp90. The sample names indicated on the figure are summarized in SI Appendix, Table S1. (A) ³¹P ENDOR spectra at different states of the Mn(II)·ATP·Hsp90 hydrolysis cycle with the pre-, HES, and posthydrolysis states in cyan, blue, and black, respectively. The gray arrows indicate the shoulder that comprises the major difference between the post- and prehydrolysis states. (B) EDNMR spectra of the different states as in A with the addition of samples prepared with ¹⁵N₅ATP. (C) As in B, with zoom on the nitrogen signals showing also the ¹⁵N hyperfine coupling.

Fig. 4.

Mn(II)–Mn(II) DEER measurements of yHsp90. The sample names indicated on the figure are summarized in SI Appendix, Table S1. (A) Mn(II)–Mn(II) DEER time traces at different states of the ATP cycle in WT yHsp90. The gray lines indicate the fit to the data after removing the background signal using the DeerAnalysis software (63) (primary data and validation of the background for the individual DEER traces are in SI Appendix, Figs. S20–S22). The DEER data were acquired with chirp pump pulses (–76, 78) (for set-up see Methods and SI Appendix, Methods and Fig. S7D). (B) Average Mn(II)–Mn(II) distance distributions obtained upon multiple repeats of sample preparation and/or measurements with the number of different sample preparations indicated (×n). The gray lines at ∼4.3 and 3.8 nm indicate the Mn(II)–Mn(II) maximum/center of the distance distribution for pre- and posthydrolysis states, the red dotted line indicates the distance between the Mg(II) ions in the closed cryo-EM structure of human Hsp90/Cdc37/Cdk4/MoO₄²⁻ complex [PDB ID code 5FWPB (33)], and the red dashed line represents the distance between 2 metal pseudoatoms coordinated by the nucleotide in the closed X-ray structure of yHsp90 (32) (see also Fig. 1A). (C) λ Values at different nucleotide-bound states; the error bars represent the SD among different samples and repeats.

Fig. 5.

Summary of our DEER results. (A) Apo state, showing the dimerization of the C-domains with the distances obtained from the Gd(III)–Gd(III) DEER on 2 mutants indicated. (B) Prehydrolysis closed conformation as found from the Mn(II)–Mn(II) DEER. (C) Posthydrolysis and HES compact conformation as found from the Mn(II)–Mn(II) DEER. The schematic structure of yHsp90 is shown in the spirit of Schulze et al. (figure 6 in ref. 17). All maxima of the distance distributions are noted on the figure. To illustrate the broader distance distribution for the ADP-bound state in C, 2 positions were drawn for the N-domains.