Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation

Abstract

Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated with neurodegenerative diseases. However, a definitive structural mechanism for its disaggregase activity has remained elusive. We determined the cryo-EM structure of wild-type Saccharomyces cerevisiae Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the 12 AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, thus explaining roles in ATP hydrolysis and protein disaggregation. Remarkably, substrate-binding pore loops line the channel in a spiral arrangement optimized for substrate transfer across the AAA+ domains, thereby establishing a continuous path for polypeptide translocation.

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Figure 1: Spiral architecture and three-tiered domain arrangement of the Hsp104 hexamer complex, determined by cryo-EM.
Figure 2: Protomer arrangement and molecular model of Hsp104.
Figure 3: Basis for the protomer spiral and NBD1-NBD2 AAA+ interaction at the hexamer seam.
Figure 4: Distinct arrangements of the NTD, MD and CTD in the hexamer.
Figure 5: Two-turn spiral arrangement of the substrate-binding tyrosine pore loops around the channel.
Figure 6: Models for cooperative disaggregation and substrate engagement by Hsp104.

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References

  1. 1

    Sanchez, Y. & Lindquist, S.L. HSP104 required for induced thermotolerance. Science 248, 1112–1115 (1990).

  2. 2

    Parsell, D.A., Kowal, A.S., Singer, M.A. & Lindquist, S. Protein disaggregation mediated by heat-shock protein Hsp104. Nature 372, 475–478 (1994).

  3. 3

    Mogk, A., Kummer, E. & Bukau, B. Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation. Front. Mol. Biosci. 2, 22 (2015).

  4. 4

    Glover, J.R. & Lindquist, S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73–82 (1998).

  5. 5

    Weibezahn, J. et al. Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB. Cell 119, 653–665 (2004).

  6. 6

    Lum, R., Tkach, J.M., Vierling, E. & Glover, J.R. Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104. J. Biol. Chem. 279, 29139–29146 (2004).

  7. 7

    Motohashi, K., Watanabe, Y., Yohda, M. & Yoshida, M. Heat-inactivated proteins are rescued by the DnaK.J-GrpE set and ClpB chaperones. Proc. Natl. Acad. Sci. USA 96, 7184–7189 (1999).

  8. 8

    Shorter, J. & Lindquist, S. Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers. Science 304, 1793–1797 (2004).

  9. 9

    Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G. & Liebman, S.W. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science 268, 880–884 (1995).

  10. 10

    Moriyama, H., Edskes, H.K. & Wickner, R.B. [URE3] prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p. Mol. Cell. Biol. 20, 8916–8922 (2000).

  11. 11

    Lo Bianco, C. et al. Hsp104 antagonizes alpha-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease. J. Clin. Invest. 118, 3087–3097 (2008).

  12. 12

    Jackrel, M.E. et al. Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events. Cell 156, 170–182 (2014).

  13. 13

    Cushman-Nick, M., Bonini, N.M. & Shorter, J. Hsp104 suppresses polyglutamine-induced degeneration post onset in a drosophila MJD/SCA3 model. PLoS Genet. 9, e1003781 (2013).

  14. 14

    Jackrel, M.E. & Shorter, J. Reversing deleterious protein aggregation with re-engineered protein disaggregases. Cell Cycle 13, 1379–1383 (2014).

  15. 15

    Rosenzweig, R. et al. ClpB N-terminal domain plays a regulatory role in protein disaggregation. Proc. Natl. Acad. Sci. USA 112, E6872–E6881 (2015).

  16. 16

    Doyle, S.M., Hoskins, J.R. & Wickner, S. DnaK chaperone-dependent disaggregation by caseinolytic peptidase B (ClpB) mutants reveals functional overlap in the N-terminal domain and nucleotide-binding domain-1 pore tyrosine. J. Biol. Chem. 287, 28470–28479 (2012).

  17. 17

    Kedzierska, S., Akoev, V., Barnett, M.E. & Zolkiewski, M. Structure and function of the middle domain of ClpB from Escherichia coli. Biochemistry 42, 14242–14248 (2003).

  18. 18

    Rosenzweig, R., Moradi, S., Zarrine-Afsar, A., Glover, J.R. & Kay, L.E. Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction. Science 339, 1080–1083 (2013).

  19. 19

    Seyffer, F. et al. Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces. Nat. Struct. Mol. Biol. 19, 1347–1355 (2012).

  20. 20

    Lee, J. et al. Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor. Proc. Natl. Acad. Sci. USA 110, 8513–8518 (2013).

  21. 21

    DeSantis, M.E. et al. Conserved distal loop residues in the Hsp104 and ClpB middle domain contact nucleotide-binding domain 2 and enable Hsp70-dependent protein disaggregation. J. Biol. Chem. 289, 848–867 (2014).

  22. 22

    Mackay, R.G., Helsen, C.W., Tkach, J.M. & Glover, J.R. The C-terminal extension of Saccharomyces cerevisiae Hsp104 plays a role in oligomer assembly. Biochemistry 47, 1918–1927 (2008).

  23. 23

    Lee, S. et al. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell 115, 229–240 (2003).

  24. 24

    Lee, S., Choi, J.M. & Tsai, F.T. Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB. Mol. Cell 25, 261–271 (2007).

  25. 25

    Wendler, P. et al. Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. Cell 131, 1366–1377 (2007).

  26. 26

    Carroni, M. et al. Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation. eLife 3, e02481 (2014).

  27. 27

    Sweeny, E.A. et al. The Hsp104 N-terminal domain enables disaggregase plasticity and potentiation. Mol. Cell 57, 836–849 (2015).

  28. 28

    Hattendorf, D.A. & Lindquist, S.L. Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants. EMBO J. 21, 12–21 (2002).

  29. 29

    Wendler, P. et al. Motor mechanism for protein threading through Hsp104. Mol. Cell 34, 81–92 (2009).

  30. 30

    DeSantis, M.E. et al. Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients. Cell 151, 778–793 (2012).

  31. 31

    Bösl, B., Grimminger, V. & Walter, S. Substrate binding to the molecular chaperone Hsp104 and its regulation by nucleotides. J. Biol. Chem. 280, 38170–38176 (2005).

  32. 32

    Tessarz, P., Mogk, A. & Bukau, B. Substrate threading through the central pore of the Hsp104 chaperone as a common mechanism for protein disaggregation and prion propagation. Mol. Microbiol. 68, 87–97 (2008).

  33. 33

    Lee, S., Sielaff, B., Lee, J. & Tsai, F.T. CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation. Proc. Natl. Acad. Sci. USA 107, 8135–8140 (2010).

  34. 34

    Parsell, D.A., Kowal, A.S. & Lindquist, S. Saccharomyces cerevisiae Hsp104 protein: purification and characterization of ATP-induced structural changes. J. Biol. Chem. 269, 4480–4487 (1994).

  35. 35

    Aguado, A., Fernández-Higuero, J.A., Cabrera, Y., Moro, F. & Muga, A. ClpB dynamics is driven by its ATPase cycle and regulated by the DnaK system and substrate proteins. Biochem. J. 466, 561–570 (2015).

  36. 36

    Werbeck, N.D., Schlee, S. & Reinstein, J. Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB. J. Mol. Biol. 378, 178–190 (2008).

  37. 37

    Mogk, A. et al. Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity. J. Biol. Chem. 278, 17615–17624 (2003).

  38. 38

    Abbas-Terki, T., Donzé, O., Briand, P.A. & Picard, D. Hsp104 interacts with Hsp90 cochaperones in respiring yeast. Mol. Cell. Biol. 21, 7569–7575 (2001).

  39. 39

    Dulle, J.E., Stein, K.C. & True, H.L. Regulation of the Hsp104 middle domain activity is critical for yeast prion propagation. PLoS One 9, e87521 (2014).

  40. 40

    Oguchi, Y. et al. A tightly regulated molecular toggle controls AAA+ disaggregase. Nat. Struct. Mol. Biol. 19, 1338–1346 (2012).

  41. 41

    Schlieker, C. et al. Substrate recognition by the AAA+ chaperone ClpB. Nat. Struct. Mol. Biol. 11, 607–615 (2004).

  42. 42

    Hinnerwisch, J., Fenton, W.A., Furtak, K.J., Farr, G.W. & Horwich, A.L. Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell 121, 1029–1041 (2005).

  43. 43

    Glynn, S.E., Martin, A., Nager, A.R., Baker, T.A. & Sauer, R.T. Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. Cell 139, 744–756 (2009).

  44. 44

    Li, T. et al. Escherichia coli ClpB is a non-processive polypeptide translocase. Biochem. J. 470, 39–52 (2015).

  45. 45

    Aubin-Tam, M.E., Olivares, A.O., Sauer, R.T., Baker, T.A. & Lang, M.J. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145, 257–267 (2011).

  46. 46

    Maillard, R.A. et al. ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145, 459–469 (2011).

  47. 47

    Olivares, A.O., Nager, A.R., Iosefson, O., Sauer, R.T. & Baker, T.A. Mechanochemical basis of protein degradation by a double-ring AAA+ machine. Nat. Struct. Mol. Biol. 21, 871–875 (2014).

  48. 48

    Guo, F., Maurizi, M.R., Esser, L. & Xia, D. Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J. Biol. Chem. 277, 46743–46752 (2002).

  49. 49

    Alexopoulos, J.A., Guarné, A. & Ortega, J. ClpP: a structurally dynamic protease regulated by AAA+ proteins. J. Struct. Biol. 179, 202–210 (2012).

  50. 50

    Haslberger, T. et al. M domains couple the ClpB threading motor with the DnaK chaperone activity. Mol. Cell 25, 247–260 (2007).

  51. 51

    Blok, N.B. et al. Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy. Proc. Natl. Acad. Sci. USA 112, E4017–E4025 (2015).

  52. 52

    Zhao, M. et al. Mechanistic insights into the recycling machine of the SNARE complex. Nature 518, 61–67 (2015).

  53. 53

    Lin, J. & Lucius, A.L. Examination of ClpB quaternary structure and linkage to nucleotide binding. Biochemistry 55, 1758–1771 (2016).

  54. 54

    Li, X., Zheng, S., Agard, D.A. & Cheng, Y. Asynchronous data acquisition and on-the-fly analysis of dose fractionated cryoEM images by UCSFImage. J. Struct. Biol. 192, 174–178 (2015).

  55. 55

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

  56. 56

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

  57. 57

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

  58. 58

    Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  59. 59

    Lander, G.C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009).

  60. 60

    Kucukelbir, A., Sigworth, F.J. & Tagare, H.D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  61. 61

    Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  62. 62

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

  63. 63

    Afonine, P.V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

  64. 64

    Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

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Acknowledgements

The authors thank J. Smith, Z. March, M. DeSantis, E. Sweeny and J. Lin for reading and discussing the manuscript; S. King for help with figures; and A. Tariq for technical assistance with Hsp104 purification. This work was supported by National Institutes of Health (NIH) grant R01GM099836 (to J.S.). A.L.Y. is supported by an American Heart Association Predoctoral fellowship; M.E.J. is supported by a Target ALS Springboard Fellowship. K.L.M. is supported by an NSF Graduate Research Fellowship (DGE-1321851). J.S. is supported by a Muscular Dystrophy Association Research Award (MDA277268), the Life Extension Foundation, the Packard Center for ALS Research at Johns Hopkins University, and Target ALS. D.R.S. is supported by NIH grants R01GM109896, R01GM077430 and R01GM110001A.

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Authors

Contributions

A.L.Y. designed experiments, performed cryo-EM sample preparation, data collection and analysis, and wrote the manuscript; S.N.G. performed cryo-EM data collection and analysis; M.E.J. purified proteins, performed biochemical analysis and edited the manuscript; K.L.M. performed biochemical analysis; M.S. supervised cryo-EM data collection; J.S. designed experiments and edited the manuscript; D.R.S. designed and supervised the study and edited the manuscript.

Corresponding author

Correspondence to Daniel R Southworth.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Functional and cryo-EM analysis of Hsp104.

(a) Purified Hsp104 hexamer is active for ATPase function, measured as the release of inorganic phosphate over 5 minutes. Value represents mean ± SEM (n = 3). (b) Purified Hsp104 is active for luciferase disaggregation. Recovered luciferase luminescence determined following incubations with Hsp104 plus (checkered bars) or minus (clear bars) equimolar Hsc70 and Hdj2. Values represent means ± SEM (n = 3). (c) Representative cryo-EM micrograph of Hsp104 hexamers. Scale bar equals 200 Å (d) 2D reference free class averages of Hsp104. The top 50 populated views are shown. (e) Gold standard FSC curve for the un-masked and masked reconstructions of the final model estimated from the FSC=0.143 criterion to be 6.54 Å and 5.64 Å, respectively. (f) 3D plot of the angular distribution for the particles that contributed to the final model. Top and side view orientations are shown with the corresponding 2D projection average. (g) Comparison of reference free class averages and 2D projections of the final 3D reconstruction. Scale bar equals 50 Å. (h) Local resolution of the 3D reconstruction determined by ResMap1 and shown from 5 Å (blue) to 7 Å (red).1 Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat Methods 11, 63–65, doi:10.1038/nmeth.2727 (2014).

Supplementary Figure 2 ClpB structure and molecular models of the Hsp104 protomers.

(a) ClpB structure labeled for reference, as previously reported1 (PDB: 1QVR), with helices indicated for the NTD (A), NBD1 large (B) and small (C) subdomains, MD (L) and NBD2 large (D) and small (E) subdomains. (b) Hsp104 molecular models for each protomer in the asymmetric hexamer with the segmented density shown at 5σ. Below are enlarged views of the nucleotide pockets with difference maps, generated by subtracting a model with no nucleotide from the cryo-EM map, shown as mesh to visualize density for bound nucleotide. AMPPNP is shown as sticks where density is observed in the pocket for the difference maps.1 Lee, S. et al. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell 115, 229–240 (2003).

Supplementary Figure 3 Conformational differences in the AAA+ domains of the individual protomers.

(a) Overlay of the backbone trace for the protomer NBDs following alignment to the P1 NBD1 large subdomain. RMSD values compared to P1 are shown and identify increasing differences from P2 to P6. (b) Enlarged view of circled region of (a) showing helices E2 and E3 for the 6 aligned protomers, highlighting conformational changes for P1 to P6. (c) Overlay of P1 and P6 protomers that interact at the hexamer interface, showing conformational changes including rotation of the NBD1 and NBD2 small subdomains.

Supplementary Figure 4 Proposed MD NBD1 cross-protomer stabilizing interactions.

Views showing model with proposed salt bridge interactions between MD-L1 and adjacent NBD1 that bridge the large and small AAA+ subdomains across nucleotide-binding pocket.

Supplementary Figure 5 Additional NBD1 pore loops line the Hsp104 channel and potentially function as substrate-binding sites.

(a) Views of the channel interface with density corresponding to putative NBD1 substrate binding surfaces shown in yellow that include flexible pore loop residues 288-298. Adjacent Tyr pore loop regions are shown as in Figure 5. Hsp104 sequence is shown with conserved charged residues indicated (asterisk).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 1010 kb)

41594_2016_BFnsmb3277_MOESM57_ESM.mov

Cryo-EM reconstruction of the Hsp104 hexamer colored by domain, as in Figure 1 (MOV 43968 kb)

Hsp104 3D reconstruction

Cryo-EM reconstruction of the Hsp104 hexamer colored by domain, as in Figure 1 (MOV 43968 kb)

41594_2016_BFnsmb3277_MOESM58_ESM.mov

Cryo-EM map showing a single segmented protomer, P4, and the corresponding molecular model in a ribbons diagram with bound nucleotides as stick models (MOV 30167 kb)

Hsp104 protomer

Cryo-EM map showing a single segmented protomer, P4, and the corresponding molecular model in a ribbons diagram with bound nucleotides as stick models (MOV 30167 kb)

41594_2016_BFnsmb3277_MOESM59_ESM.mov

View of the Hsp104 hexamer from inside the channel. Density corresponding to the substrate binding pore loops, colored by protomer as in Figure 2 and 5, is shown arranged in a two turn spiral (MOV 186300 kb)

Hsp104 substrate-binding pore loops

View of the Hsp104 hexamer from inside the channel. Density corresponding to the substrate binding pore loops, colored by protomer as in Figure 2 and 5, is shown arranged in a two turn spiral (MOV 186300 kb)

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Yokom, A., Gates, S., Jackrel, M. et al. Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation. Nat Struct Mol Biol 23, 830–837 (2016). https://doi.org/10.1038/nsmb.3277

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