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

Nature Structural & Molecular Biology volume 23, pages 1020–1028 (2016)Cite this article

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Abstract

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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References

  1. Zhao, R. et al. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120, 715–727 (2005).

    Article  CAS  Google Scholar 

  2. Young, J.C., Moarefi, I. & Hartl, F.U. Hsp90: a specialized but essential protein-folding tool. J. Cell Biol. 154, 267–273 (2001).

    Article  CAS  Google Scholar 

  3. Picard, D. Heat-shock protein 90, a chaperone for folding and regulation. Cell. Mol. Life Sci. 59, 1640–1648 (2002).

    Article  CAS  Google Scholar 

  4. McClellan, A.J. et al. Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches. Cell 131, 121–135 (2007).

    Article  CAS  Google Scholar 

  5. Röhl, A., Rohrberg, J. & Buchner, J. The chaperone Hsp90: changing partners for demanding clients. Trends Biochem. Sci. 38, 253–262 (2013).

    Article  Google Scholar 

  6. Mayer, M.P. & Le Breton, L. Hsp90: breaking the symmetry. Mol. Cell 58, 8–20 (2015).

    Article  CAS  Google Scholar 

  7. Taipale, M., Jarosz, D.F. & Lindquist, S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. Cell Biol. 11, 515–528 (2010).

    Article  CAS  Google Scholar 

  8. Prodromou, C. et al. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90, 65–75 (1997).

    Article  CAS  Google Scholar 

  9. Nemoto, T., Ohara-Nemoto, Y., Ota, M., Takagi, T. & Yokoyama, K. Mechanism of dimer formation of the 90-kDa heat-shock protein. Eur. J. Biochem. 233, 1–8 (1995).

    Article  CAS  Google Scholar 

  10. Bose, S., Weikl, T., Bügl, H. & Buchner, J. Chaperone function of Hsp90-associated proteins. Science 274, 1715–1717 (1996).

    Article  CAS  Google Scholar 

  11. Wegele, H., Muschler, P., Bunck, M., Reinstein, J. & Buchner, J. Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90. J. Biol. Chem. 278, 39303–39310 (2003).

    Article  CAS  Google Scholar 

  12. Prodromou, C. et al. The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains. EMBO J. 19, 4383–4392 (2000).

    Article  CAS  Google Scholar 

  13. Smith, D.F. Tetratricopeptide repeat cochaperones in steroid receptor complexes. Cell Stress Chaperones 9, 109–121 (2004).

    Article  CAS  Google Scholar 

  14. Panaretou, B. et al. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J. 17, 4829–4836 (1998).

    Article  CAS  Google Scholar 

  15. Obermann, W.M., Sondermann, H., Russo, A.A., Pavletich, N.P. & Hartl, F.U. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis. J. Cell Biol. 143, 901–910 (1998).

    Article  CAS  Google Scholar 

  16. Mishra, P. & Bolon, D.N. Designed Hsp90 heterodimers reveal an asymmetric ATPase-driven mechanism in vivo. Mol. Cell 53, 344–350 (2014).

    Article  CAS  Google Scholar 

  17. Shiau, A.K., Harris, S.F., Southworth, D.R. & Agard, D.A. Structural analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements. Cell 127, 329–340 (2006).

    Article  CAS  Google Scholar 

  18. Ali, M.M. et al. Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440, 1013–1017 (2006).

    Article  CAS  Google Scholar 

  19. Hessling, M., Richter, K. & Buchner, J. Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90. Nat. Struct. Mol. Biol. 16, 287–293 (2009).

    Article  CAS  Google Scholar 

  20. Weikl, T. et al. C-terminal regions of Hsp90 are important for trapping the nucleotide during the ATPase cycle. J. Mol. Biol. 303, 583–592 (2000).

    Article  CAS  Google Scholar 

  21. Mickler, M., Hessling, M., Ratzke, C., Buchner, J. & Hugel, T. The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis. Nat. Struct. Mol. Biol. 16, 281–286 (2009).

    Article  CAS  Google Scholar 

  22. Krukenberg, K.A., Förster, F., Rice, L.M., Sali, A. & Agard, D.A. Multiple conformations of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90. Structure 16, 755–765 (2008).

    Article  CAS  Google Scholar 

  23. Richter, K., Reinstein, J. & Buchner, J. N-terminal residues regulate the catalytic efficiency of the Hsp90 ATPase cycle. J. Biol. Chem. 277, 44905–44910 (2002).

    Article  CAS  Google Scholar 

  24. Nathan, D.F. & Lindquist, S. Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase. Mol. Cell. Biol. 15, 3917–3925 (1995).

    Article  CAS  Google Scholar 

  25. Vaughan, C.K., Piper, P.W., Pearl, L.H. & Prodromou, C. A common conformationally coupled ATPase mechanism for yeast and human cytoplasmic HSP90s. FEBS J. 276, 199–209 (2009).

    Article  CAS  Google Scholar 

  26. Hubert, D.A., He, Y., McNulty, B.C., Tornero, P. & Dangl, J.L. Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation. Proc. Natl. Acad. Sci. USA 106, 9556–9563 (2009).

    Article  CAS  Google Scholar 

  27. Meyer, P. et al. Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol. Cell 11, 647–658 (2003).

    Article  CAS  Google Scholar 

  28. Retzlaff, M. et al. Asymmetric activation of the hsp90 dimer by its cochaperone aha1. Mol. Cell 37, 344–354 (2010).

    Article  CAS  Google Scholar 

  29. Koulov, A.V. et al. Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis. Mol. Biol. Cell 21, 871–884 (2010).

    Article  CAS  Google Scholar 

  30. Cunningham, C.N., Southworth, D.R., Krukenberg, K.A. & Agard, D.A. The conserved arginine 380 of Hsp90 is not a catalytic residue, but stabilizes the closed conformation required for ATP hydrolysis. Protein Sci. 21, 1162–1171 (2012).

    Article  CAS  Google Scholar 

  31. Lorenz, O.R. et al. Modulation of the Hsp90 chaperone cycle by a stringent client protein. Mol. Cell 53, 941–953 (2014).

    Article  CAS  Google Scholar 

  32. Li, J., Richter, K., Reinstein, J. & Buchner, J. Integration of the accelerator Aha1 in the Hsp90 co-chaperone cycle. Nat. Struct. Mol. Biol. 20, 326–331 (2013).

    Article  Google Scholar 

  33. Li, J., Richter, K. & Buchner, J. Mixed Hsp90–cochaperone complexes are important for the progression of the reaction cycle. Nat. Struct. Mol. Biol. 18, 61–66 (2011).

    Article  Google Scholar 

  34. Richter, K., Walter, S. & Buchner, J. The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle. J. Mol. Biol. 342, 1403–1413 (2004).

    Article  CAS  Google Scholar 

  35. Toogun, O.A., Dezwaan, D.C. & Freeman, B.C. The hsp90 molecular chaperone modulates multiple telomerase activities. Mol. Cell. Biol. 28, 457–467 (2008).

    Article  CAS  Google Scholar 

  36. Echtenkamp, F.J. et al. Global functional map of the p23 molecular chaperone reveals an extensive cellular network. Mol. Cell 43, 229–241 (2011).

    Article  CAS  Google Scholar 

  37. Mimnaugh, E.G., Worland, P.J., Whitesell, L. & Neckers, L.M. Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase. J. Biol. Chem. 270, 28654–28659 (1995).

    Article  CAS  Google Scholar 

  38. Xu, Y. & Lindquist, S. Heat-shock protein hsp90 governs the activity of pp60v-src kinase. Proc. Natl. Acad. Sci. USA 90, 7074–7078 (1993).

    Article  CAS  Google Scholar 

  39. Johnson, J.L., Halas, A. & Flom, G. Nucleotide-dependent interaction of Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1, Cpr6, and Sba1. Mol. Cell. Biol. 27, 768–776 (2007).

    Article  CAS  Google Scholar 

  40. Hawle, P. et al. The middle domain of Hsp90 acts as a discriminator between different types of client proteins. Mol. Cell. Biol. 26, 8385–8395 (2006).

    Article  CAS  Google Scholar 

  41. Richter, K. et al. Intrinsic inhibition of the Hsp90 ATPase activity. J. Biol. Chem. 281, 11301–11311 (2006).

    Article  CAS  Google Scholar 

  42. Zurawska, A. et al. Mutations that increase both Hsp90 ATPase activity in vitro and Hsp90 drug resistance in vivo. Biochim. Biophys. Acta 1803, 575–583 (2010).

    Article  CAS  Google Scholar 

  43. Tsutsumi, S. et al. Charged linker sequence modulates eukaryotic heat shock protein 90 (Hsp90) chaperone activity. Proc. Natl. Acad. Sci. USA 109, 2937–2942 (2012).

    Article  CAS  Google Scholar 

  44. Soroka, J. et al. Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation. Mol. Cell 45, 517–528 (2012).

    Article  CAS  Google Scholar 

  45. Mollapour, M. & Neckers, L. Post-translational modifications of Hsp90 and their contributions to chaperone regulation. Biochim. Biophys. Acta- 1823, 648–655 (2012).

    Article  CAS  Google Scholar 

  46. Buchner, J., Weikl, T., Bügl, H., Pirkl, F. & Bose, S. Purification of Hsp90 partner proteins Hop/p60, p23, and FKBP52. Methods Enzymol. 290, 418–429 (1998).

    Article  CAS  Google Scholar 

  47. Richter, K., Muschler, P., Hainzl, O. & Buchner, J. Coordinated ATP hydrolysis by the Hsp90 dimer. J. Biol. Chem. 276, 33689–33696 (2001).

    Article  CAS  Google Scholar 

  48. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

  49. Vranken, W.F. et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696 (2005).

    Article  CAS  Google Scholar 

  50. Svergun, D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).

    Article  CAS  Google Scholar 

  51. Stafford, W.F. III. Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal. Biochem. 203, 295–301 (1992).

    Article  CAS  Google Scholar 

  52. Hayes, D.B. & Stafford, W.F. SEDVIEW, real-time sedimentation analysis. Macromol. Biosci. 10, 731–735 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

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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Author notes

  1. Bettina K Zierer, Martin Rübbelke and Franziska Tippel: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Center for Integrated Protein Science Munich, Technische Universität München, Garching, Germany

    Bettina K Zierer, Martin Rübbelke, Franziska Tippel, Tobias Madl, Florian H Schopf, Daniel A Rutz, Klaus Richter, Michael Sattler & Johannes Buchner

  2. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany

    Martin Rübbelke, Tobias Madl & Michael Sattler

  3. Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Graz, Austria

    Tobias Madl

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  1. Bettina K Zierer
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Contributions

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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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

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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). https://doi.org/10.1038/nsmb.3305

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