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Importance of cycle timing for the function of the molecular chaperone Hsp90


Hsp90 couples ATP hydrolysis to large conformational changes essential for activation of client proteins. The structural transitions involve dimerization of the N-terminal domains and formation of 'closed states' involving the N-terminal and middle domains. Here, we used Hsp90 mutants that modulate ATPase activity and biological function as probes to address the importance of conformational cycling for Hsp90 activity. We found no correlation between the speed of ATP turnover and the in vivo activity of Hsp90: some mutants with almost normal ATPase activity were lethal, and some mutants with lower or undetectable ATPase activity were viable. Our analysis showed that it is crucial for Hsp90 to attain and spend time in certain conformational states: a certain dwell time in open states is required for optimal processing of client proteins, whereas a prolonged population of closed states has negative effects. Thus, the timing of conformational transitions is crucial for Hsp90 function and not cycle speed.

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Figure 1: Localization and effects of mutations on ATPase activities.
Figure 2: SAXS analysis of Hsp90 variants.
Figure 3: NMR analysis of N-domain mutants.
Figure 4: N-terminal closing of the different Hsp90 variants.
Figure 5: Binding of the cochaperone p23 (Sba1) to the Hsp90 variants.
Figure 6: Influence of Hsp90 variants in vivo.
Figure 7: Importance of cycle timing in the function of Hsp90.

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We acknowledge C. Göbl and C. Hartlmüller for help with the SAXS measurements, B. Tremmel for help with protein expression and purification, J. Soroka and J. Reinstein for inspiring discussions and comments on the manuscript and S. Lindquist (Whitehead Institute) for providing reagents. F.T. acknowledges a scholarship from the Studienstiftung des deutschen Volkes. This work was supported by the Bavarian Ministry of Sciences, Research and the Arts (Bavarian Molecular Biosystems Research Network, to T.M.), the Austrian Academy of Sciences (APART-fellowship to T.M.), the Austrian Science Fund (grant no. FWF: P28854 to T.M.), the Deutsche Forschungsgemeinschaft (grant no. SFB1035 to J.B. and M.S.) and the Emmy Noether program (grant no. MA 5703/1-1 to T.M.).

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Authors and Affiliations



B.K.Z. and F.T. performed experiments and data analysis. M.R. recorded and analyzed NMR data. T.M. performed and analyzed SAXS measurements. F.H.S. generated Hsp90-knockout strains and performed yeast tetrad analysis. K.R. performed the AUC experiments and analyzed data, J.B. designed experiments. B.K.Z., K.R., M.S. and J.B. wrote the manuscript. D.A.R. provided ATTO488-labeled GR and performed AUC runs.

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Correspondence to Johannes Buchner.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vivo control experiments.

(a) Test for plasmid loss. Yeast cells were streaked out on media lacking uracil after 5’FOA shuffling. As control non shuffled cells expressing Hsp90 wt (pKAT6) were used. (b) Tetrad analysis was performed with p415-GPD-HSP82E33A or the empty vector (ev) as control. The resulted Hsp90 double knock-out strain is complemented by Hsp90 variant E33A (left panel) whereas the empty vector p415GPD was not able to support growth of the double knock-out as always one spore is inviable (right panel). The distributions of the kanMX cassette show tetra type (TT) distribution in the E33A complemented strain, always one out of four spores must carry a genomic double knock-out of Hsp90. In the vector control only two spores are G418 resistant confirming that the deletion of both Hsp90 alleles is lethal when Hsp90 is not provided by a plasmid. (c) 5’FOA Shuffling approach was carried out with different yeast shuffling strains and under GPD- and endogenous promotor. In all strains Hsp90 variant E33A supports yeast cell growth.

Supplementary Figure 2 SAXS scattering curves.

Experimental X-ray scattering data of Hsp90 versions recorded at different sample concentrations. Both the s, and I(s) axes are shown in a logarithmic representation. The angular ranges from 0.0012 - 0.4 nm-1 are compared.

Supplementary Figure 3 1H,15N-HSQC spectra of the Hsp90 N-domain mutants.

1H,15N-HSQC spectra of the indicated mutant (red) are superposed with spectra of the wildtype (black) of the N-terminal domain of yeast Hsp90. Negative peaks are plotted in orange and grey respectively. Examples of strongly shifting peaks are highlighted in boxes.

Supplementary Figure 4 Chemical-shift-perturbation plots of the Hsp90 N-domain mutants.

Chemical shift perturbation of the 1H,15N-HSQC spectra from Supplementary Figure 3 between the indicated mutant and the wildtype of the N-terminal domain of yeast Hsp90. Negative chemical shifts indicate shifting residues that could not be assigned in the complex. Residues which are not assigned in the wildtype are indicated by gaps. Chemical shift changes larger than 0.15 ppm are indicated on top of the bars. The red lines indicate the cut-off values used in Figure 2.

Supplementary Figure 5 1H,15N-HSQC spectra of the Hsp90 N-domain mutants in complex with nucleotide.

1H,15N-HSQC spectra of the indicated variant of the N-terminal domain of yeast Hsp90 with (red) and without (black) the indicated nucleotide are superposed. Negative peaks are plotted in orange and grey respectively. Examples of strongly shifting peaks are highlighted in boxes.

Supplementary Figure 6 Fluorescence spectra of the N- and M-domain labeled Hsp90 heterocomplex in the absence or presence of nucleotides.

The inset indicates the subunit exchange of the Hsp90 variants. Decrease in donor channel and increase in acceptor channel fluorescence were monitored and indicate FRET (inset). Following fluorescence spectra were recorded: donor only (grey), Hsp90 hetero-complex without nucleotide (black), in the presence of ATP (red) and ATPγS (blue).

Supplementary Figure 7 Hsp90-Aha1 interaction in vitro.

(a) The binding of the labeled co-chaperone Aha1* to different Hsp90 mutants in the presence of ATP monitored by analytical ultracentrifugation with fluorescence detection and derived from dc/dt plots (left panel). Following color code is used: Aha1* in brown, Aha1* in complex with: wt in black, A107N in yellow, Δ8 in blue, T22I in green, R346S in purple, R380A in grey, E33A in red and D79N in light blue. The areas of the Hsp90-Aha1* complex peaks in presence of 2 mM ATP (right panel). Error bars indicate standard error of the fit. (b) N-terminal dimerization stability of the Hsp90 variants in presence of ATP and Aha1. FRET chase experiments were performed with preformed Hsp90 FRET-complexes in the presence of ATP and the co-chaperone Aha1. The chase was induced by addition of 20-fold excess of the unlabeled Hsp90 D79N and the disruption of complex was monitored by following the decrease in acceptor fluorescence. Apparent half-lives (t1/2) were derived from a non-linear fit of the acceptor signal changes. Means of technical replicates are shown. Error bars indicate s.d. (n value=3).

Supplementary Figure 8 Nucleotide-dependent Hsp90-p23 and Hsp90-GR interactions in vitro.

Hsp90 variants were titrated with increasing amounts of fluorescein-labeled p23*. The binding of p23 to different Hsp90 mutants in the presence of (a) 2 mM ATPγS, (b) 2 mM ATP and (c) without nucleotide were monitored by analytical ultracentrifugation with a fluorescence detection unit and derived from dc/dt plots. (d) The binding of labeled GR* to different Hsp90 mutants in the absence of nucleotide were monitored by analytical ultracentrifugation and derived from dc/dt plots. Following color code is used: GR* in brown, GR* in complex with: wt in black, A107N in yellow, Δ8 in blue, T22I in green, R346S in purple, R380A in grey, E33A in red and D79N in light blue.

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Supplementary Figures 1–8 and Supplementary Tables 1–4 (PDF 1828 kb)

Supplementary Data Set 1

Uncropped Western Blot data (PDF 149 kb)

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Zierer, B., Rübbelke, M., Tippel, F. et al. Importance of cycle timing for the function of the molecular chaperone Hsp90. Nat Struct Mol Biol 23, 1020–1028 (2016).

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