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:

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation

Nature Structural & Molecular Biology volume 11, pages 1128–1133 (2004)Cite this article

Abstract

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Structural comparison of wild-type (WT) and E461K GroEL.
Figure 2: Calculation of the energy of the ring interface in wild-type and mutant GroEL as a function of angle of rotation relative to the position found in wild-type GroEL.
Figure 3: Initial rate of ATPase activity as a function of ATP concentration.
Figure 4: Temperature sensitivity of GroEL E461K–GroES complexes in the presence of 4 mM ATP observed by negative-stain EM.
Figure 5: Allosteric communication in wild-type (WT) and E461K GroEL.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Sigler, P.B. et al. Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581–608 (1998).

    Article  CAS  Google Scholar 

  2. Brinker, A. et al. Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223–233 (2001).

    Article  CAS  Google Scholar 

  3. Saibil, H.R. & Ranson, N.A. The chaperonin folding machine. Trends Biochem. Sci. 27, 627–632 (2002).

    Article  CAS  Google Scholar 

  4. 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  Google Scholar 

  5. Rye, H.S. et al. GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings. Cell 97, 325–338 (1999).

    Article  CAS  Google Scholar 

  6. Gray, T.E. & Fersht, A.R. Cooperativity in ATP hydrolysis by GroEL is increased by GroES. FEBS Lett. 292, 254–258 (1991).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Burston, S.G., Ranson, N.A. & Clarke, A.R. The origins and consequences of asymmetry in the chaperonin reaction cycle. J. Mol. Biol. 249, 138–152 (1995).

    Article  CAS  Google Scholar 

  9. Roseman, A.M., Chen, S., White, H., Braig, K. & Saibil, H.R. The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell 87, 241–251 (1996).

    Article  CAS  Google Scholar 

  10. Rye, H.S. et al. Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL. Nature 388, 792–798 (1997).

    Article  CAS  Google Scholar 

  11. Ranson, N.A. et al. ATP-bound states of GroEL captured by cryo-electron microscopy. Cell 107, 869–879 (2001).

    Article  CAS  Google Scholar 

  12. Ditzel, L. et al. Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT. Cell 93, 125–138 (1998).

    Article  CAS  Google Scholar 

  13. Horwich, A.L., Low, K.B., Fenton, W.A., Hirshfield, I.N. & Furtak, K. Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell 74, 909–917 (1993).

    Article  CAS  Google Scholar 

  14. Fenton, W.A., Kashi, Y., Furtak, K. & Horwich, A.L. Residues in chaperonin GroEL required for polypeptide binding and release. Nature 371, 614–661 (1994).

    Article  CAS  Google Scholar 

  15. Sot, B., Galan, A., Valpuesta, J.M., Bertrand, S. & Muga, A. Salt bridges at the inter-ring interface regulate the thermostat of GroEL. J. Biol. Chem. 277, 34024–34029 (2002).

    Article  CAS  Google Scholar 

  16. Yifrach, O. & Horovitz, A. Two lines of allosteric communication in the oligomeric chaperonin GroEL are revealed by the single mutation Arg196→Ala. J. Mol. Biol. 243, 397–401 (1994).

    Article  CAS  Google Scholar 

  17. Weissman, J.S. et al. Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES. Cell 83, 577–587 (1995).

    Article  CAS  Google Scholar 

  18. Ma, J., Sigler, P.B., Xu, Z. & Karplus, M. A dynamic model for the allosteric mechanism of GroEL. J. Mol. Biol. 302, 303–313 (2000).

    Article  CAS  Google Scholar 

  19. Kafri, G. & Horovitz, A. Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1. J. Mol. Biol. 326, 981–987 (2003).

    Article  CAS  Google Scholar 

  20. Braig, K. et al. The crystal structure of the bacterial chaperonin GroEL at 2.8 Å. Nature 371, 578–586 (1994).

    Article  CAS  Google Scholar 

  21. Horovitz, A., Bochkareva, E.S., Kovalenko, O. & Girshovich, A.S. Mutation Ala2→Ser destabilizes intersubunit interactions in the molecular chaperone GroEL. J. Mol. Biol. 231, 58–64 (1993).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Crowther, R.A., Henderson, R. & Smith, J.M. MRC image processing programs. J. Struct. Biol. 116, 9–16 (1996).

    Article  CAS  Google Scholar 

  24. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  Google Scholar 

  25. Braig, K., Adams, P.D. & Brunger, A.T. Conformational variability in the refined structure of the chaperonin GroEL at 2.8 Å resolution. Nat. Struct. Biol. 2, 1083–1094 (1995).

    Article  CAS  Google Scholar 

  26. Brunger, A.T. & Karplus, M. Polar hydrogen positions in proteins—empirical energy placement and neutron diffraction comparison. Proteins 4, 148–156 (1988).

    Article  CAS  Google Scholar 

  27. Brooks B.R. et al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comp. Chem. 4, 187–217 (1983).

    Article  CAS  Google Scholar 

  28. MacKerell, A.D. Jr. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Westlake, D. Houldershaw and S. Terrill for computing support, W. Fenton and M. Karplus for advice and discussion, M. Jaffer and W. Williams for help with particle counting, and The Wellcome Trust and the Howard Hughes Medical Institute for financial support.

Author information

Author notes

  1. Robert B Best

    Present address: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892-0520, USA

  2. Shaoxia Chen & Alan M Roseman

    Present address: Medical Research Council Laboratory of Molecular Biology, MRC Centre, Cambridge, CB2 1QH, UK

Authors and Affiliations

  1. Electron Microscope Unit and Department of Chemistry, University of Cape Town, Rondebosch, South Africa

    B Trevor Sewell & Robert B Best

  2. Crystallography Department, Birkbeck College, London, WC1E 7HX, UK

    Shaoxia Chen, Alan M Roseman & Helen R Saibil

  3. Department of Genetics and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, 06510, Connecticut, USA

    George W Farr & Arthur L Horwich

  4. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892-0520, USA

    Shaoxia Chen & Alan M Roseman

  5. Medical Research Council Laboratory of Molecular Biology, MRC Centre, Cambridge, CB2 1QH, UK

    Robert B Best

Authors
  1. B Trevor Sewell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert B Best
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaoxia Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alan M Roseman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. George W Farr
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arthur L Horwich
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Helen R Saibil
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Helen R Saibil.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sewell, B., Best, R., Chen, S. et al. A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation. Nat Struct Mol Biol 11, 1128–1133 (2004). https://doi.org/10.1038/nsmb844

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb844

This article is cited by

Search

Advanced 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