Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins

Abstract

During and after protein translation, molecular chaperones require ATP hydrolysis to favor the native folding of their substrates and, under stress, to avoid aggregation and revert misfolding. Why do some chaperones need ATP, and what are the consequences of the energy contributed by the ATPase cycle? Here, we used biochemical assays and physical modeling to show that the bacterial chaperones GroEL (Hsp60) and DnaK (Hsp70) both use part of the energy from ATP hydrolysis to restore the native state of their substrates, even under denaturing conditions in which the native state is thermodynamically unstable. Consistently with thermodynamics, upon exhaustion of ATP, the metastable native chaperone products spontaneously revert to their equilibrium non-native states. In the presence of ATPase chaperones, some proteins may thus behave as open ATP-driven, nonequilibrium systems whose fate is only partially determined by equilibrium thermodynamics.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Free-energy landscape of MDH at 37 °C and schematic action of chaperones.
Fig. 2: ATP-dependent nonequilibrium GroELS recovery and maintenance of MDH activity in denaturing conditions.
Fig. 3: Results from the model.
Fig. 4: ATP-driven catalytic action of substoichiometric amounts of GroELS on MDH at 37 °C.
Fig. 5: GroELS converts the energy of ATP into a nonequilibrium stabilization of the native state of MDH.

Similar content being viewed by others

References

  1. Anfinsen, C. B., Haber, E., Sela, M. & White, F. H. Jr. The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proc. Natl. Acad. Sci. USA 47, 1309–1314 (1961).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Onuchic, J. N. & Wolynes, P. G. Theory of protein folding. Curr. Opin. Struct. Biol. 14, 70–75 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Guo, Z. Y. & Thirumalai, D. Kinetics of protein-folding: nucleation mechanism, time scales, and pathways. Biopolymers 36, 83–102 (1995).

    Article  CAS  Google Scholar 

  4. Knowles, T. P. J., Vendruscolo, M. & Dobson, C. M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15, 384–396 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Finka, A., Mattoo, R. U. H. & Goloubinoff, P. Experimental milestones in the discovery of molecular chaperones as polypeptide unfolding enzymes. Annu. Rev. Biochem. 85, 715–742 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Gat-Yablonski, G. et al. Quantitative proteomics of rat livers shows that unrestricted feeding is stressful for proteostasis with implications on life span. Aging (Albany NY) 8, 1735–1758 (2016).

    Article  CAS  Google Scholar 

  7. Goloubinoff, P., Christeller, J. T., Gatenby, A. A. & Lorimer, G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature 342, 884–889 (1989).

    Article  CAS  PubMed  Google Scholar 

  8. Jakob, U., Gaestel, M., Engel, K. & Buchner, J. Small heat shock proteins are molecular chaperones. J. Biol. Chem. 268, 1517–1520 (1993).

    CAS  PubMed  Google Scholar 

  9. Koldewey, P., Stull, F., Horowitz, S., Martin, R. & Bardwell, J. C. A. Forces driving chaperone action. Cell 166, 369–379 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Frank, G. A. et al. Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations. Proc. Natl. Acad. Sci. USA 107, 6270–6274 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  11. De Los Rios, P. & Barducci, A. Hsp70 chaperones are non-equilibrium machines that achieve ultra-affinity by energy consumption. eLife 3, e02218 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Barducci, A. & De Los Rios, P. Non-equilibrium conformational dynamics in the function of molecular chaperones. Curr. Opin. Struct. Biol. 30, 161–169 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Goloubinoff, P., Gatenby, A. A. & Lorimer, G. H. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 337, 44–47 (1989).

    Article  CAS  PubMed  Google Scholar 

  14. Hayer-Hartl, M., Bracher, A. & Hartl, F. U. The GroEL-GroES chaperonin machine: a nano-cage for protein folding. Trends Biochem. Sci. 41, 62–76 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Hartman, D. J., Surin, B. P., Dixon, N. E., Hoogenraad, N. J. & Høj, P. B. Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro. Proc. Natl. Acad. Sci. USA 90, 2276–2280 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dobson, C. M. & Karplus, M. The fundamentals of protein folding: bringing together theory and experiment. Curr. Opin. Struct. Biol. 9, 92–101 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Peralta, D., Hartman, D. J., Hoogenraad, N. J. & Høj, P. B. Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES. FEBS Lett. 339, 45–49 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Buchner, J. et al. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30, 1586–1591 (1991).

    Article  CAS  PubMed  Google Scholar 

  19. Moran, U., Phillips, R. & Milo, R. SnapShot: key numbers in biology. Cell 141, 1262–1262.e1 (2010).

    Article  PubMed  Google Scholar 

  20. Priya, S. et al. GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP. Proc. Natl. Acad. Sci. USA 110, 7199–7204 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Horst, R. et al. Direct NMR observation of a substrate protein bound to the chaperonin GroEL. Proc. Natl. Acad. Sci. USA 102, 12748–12753 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lin, Z., Madan, D. & Rye, H. S. GroEL stimulates protein folding through forced unfolding. Nat. Struct. Mol. Biol. 15, 303–311 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sharma, S. et al. Monitoring protein conformation along the pathway of chaperonin-assisted folding. Cell 133, 142–153 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Libich, D. S., Tugarinov, V. & Clore, G. M. Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR. Proc. Natl. Acad. Sci. USA 112, 8817–8823 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Weaver, J. et al. GroEL actively stimulates folding of the endogenous substrate protein PepQ. Nat. Commun. 8, 15934 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. van der Vaart, A., Ma, J. & Karplus, M. The unfolding action of GroEL on a protein substrate. Biophys. J. 87, 562–573 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Stan, G., Lorimer, G. H., Thirumalai, D. & Brooks, B. R. Coupling between allosteric transitions in GroEL and assisted folding of a substrate protein. Proc. Natl. Acad. Sci. USA 104, 8803–8808 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sharma, S. K., De Los Rios, P., Christen, P., Lustig, A. & Goloubinoff, P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat. Chem. Biol. 6, 914–920 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Kellner, R. et al. Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein. Proc. Natl. Acad. Sci. USA 111, 13355–13360 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lee, J. H. et al. Heterogeneous binding of the SH3 client protein to the DnaK molecular chaperone. Proc. Natl. Acad. Sci. USA 112, E4206–E4215 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sekhar, A., Rosenzweig, R., Bouvignies, G. & Kay, L. E. Mapping the conformation of a client protein through the Hsp70 functional cycle. Proc. Natl. Acad. Sci. USA 112, 10395–10400 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tehver, R. & Thirumalai, D. Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation. J. Mol. Biol. 377, 1279–1295 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Santra, M., Farrell, D. W. & Dill, K. A. Bacterial proteostasis balances energy and chaperone utilization efficiently. Proc. Natl. Acad. Sci. USA 114, E2654–E2661 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Priya, S., Sharma, S. K. & Goloubinoff, P. Molecular chaperones as enzymes that catalytically unfold misfolded polypeptides. FEBS Lett. 587, 1981–1987 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Yifrach, O. & Horovitz, A. Nested cooperativity in the ATPase activity of the oligomeric chaperonin GroEL. Biochemistry 34, 5303–5308 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Corrales, F. J. & Fersht, A. R. Toward a mechanism for GroEL.GroES chaperone activity: an ATPase-gated and -pulsed folding and annealing cage. Proc. Natl. Acad. Sci. USA 93, 4509–4512 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Todd, M. J., Lorimer, G. H. & Thirumalai, D. Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism. Proc. Natl. Acad. Sci. USA 93, 4030–4035 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Doyle, S. M. & Wickner, S. Hsp104 and ClpB: protein disaggregating machines. Trends Biochem. Sci. 34, 40–48 (2009).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  40. Langer, T. et al. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 356, 683–689 (1992).

    Article  CAS  PubMed  Google Scholar 

  41. Buchberger, A., Schröder, H., Hesterkamp, T., Schönfeld, H. J. & Bukau, B. Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding. J. Mol. Biol. 261, 328–333 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Sharma, S. K., De Los Rios, P. & Goloubinoff, P. Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate. Proteins 79, 1991–1998 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Rajkowitsch, L. & Schroeder, R. Dissecting RNA chaperone activity. RNA 13, 2053–2060 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Quan, S. et al. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat. Struct. Mol. Biol. 18, 262–269 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lindquist, S. Protein folding sculpting evolutionary change. Cold Spring Harb. Symp. Quant. Biol. 74, 103–108 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Tokuriki, N. & Tawfik, D. S. Chaperonin overexpression promotes genetic variation and enzyme evolution. Nature 459, 668–673 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Bhaskaran, H. & Russell, R. Kinetic redistribution of native and misfolded RNAs by a DEAD-box chaperone. Nature 449, 1014–1018 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yang, Q., Fairman, M. E. & Jankowsky, E. DEAD-box-protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values. J. Mol. Biol. 368, 1087–1100 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bershtein, S., Mu, W., Serohijos, A. W. R., Zhou, J. & Shakhnovich, E. I. Protein quality control acts on folding intermediates to shape the effects of mutations on organismal fitness. Mol. Cell 49, 133–144 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Schrödinger, E. What is life? (Cambridge University Press, Cambridge, 1945).

    Google Scholar 

  51. Ranson, N. A., Dunster, N. J., Burston, S. G. & Clarke, A. R. Chaperonins can catalyse the reversal of early aggregation steps when a protein misfolds. J. Mol. Biol. 250, 581–586 (1995).

  52. Török, Z. et al. Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions. Proc. Natl. Acad. Sci. USA 94, 2192–2197 (1997).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Gur, E. et al. The Escherichia coli DjlA and CbpA proteins can substitute for DnaJ in DnaK-mediated protein disaggregation. J. Bacteriol. 186, 7236–7242 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Woo, K. M., Kim, K. I., Goldberg, A. L., Ha, D. B. & Chung, C. H. The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase. J. Biol. Chem. 267, 20429–20434 (1992).

    CAS  PubMed  Google Scholar 

  55. Diamant, S., Azem, A., Weiss, C. & Goloubinoff, P. Increased efficiency of GroE-assisted protein folding by manganese ions. J. Biol. Chem. 270, 28387–28391 (1995).

    Article  CAS  PubMed  Google Scholar 

  56. Finka, A. & Goloubinoff, P. Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis. Cell Stress Chaperon. 18, 591–605 (2013).

    Article  CAS  Google Scholar 

  57. Todd, M. J. & Lorimer, G. H. Stability of the asymmetric Escherichia coli chaperonin complex: guanidine chloride causes rapid dissociation. J. Biol. Chem. 270, 5388–5394 (1995).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank summer-project students P. Inns and I. Koog for setting up the citrate synthase enzymatic assay and performing key preliminary and control chaperone assays. This project was financed in part by Swiss National Science Foundation grants 140512/1 and 31003A_156948, and Swiss State Secretariat for Education Research and Innovation grant C15.0042 to P.G. and B.F., and by Swiss National Science Foundation grant 200020_163042 to P.D.L.R. and A.S.S. A.B. acknowledges support from the French Agence Nationale de la Recherche (ANR), under grant ANR-14-ACHN-0016. We thank H.-J. Schönfeld (F. Hoffmann-La Roche) for providing GrpE.

Author information

Authors and Affiliations

Authors

Contributions

P.G., A.B. and P.D.L.R. conceived the study. P.G. and B.F. performed the experiments. P.D.L.R., A.S.S. and A.B. developed the model. P.D.L.R. and A.S.S. performed the calculations. All authors contributed to the writing of the work.

Corresponding authors

Correspondence to Pierre Goloubinoff or Paolo De Los Rios.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Note

Life Sciences Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Goloubinoff, P., Sassi, A.S., Fauvet, B. et al. Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins. Nat Chem Biol 14, 388–395 (2018). https://doi.org/10.1038/s41589-018-0013-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0013-8

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing