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Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis

Nature Structural & Molecular Biology volume 12, pages 233–237 (2005)Cite this article

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Abstract

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTα and CCTδ subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.

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Figure 1: Steps in the alignment of images of 8g antibody-bound CCT particles illustrated for apo CCT and particle classification.
Figure 2: Symmetry analysis of class averages of 8g monoclonal antibody-bound CCT particles in the presence of different concentrations of ATP.
Figure 3: Average power spectra of the rotational correlation plots corresponding to the class averages at different concentrations of ATP.
Figure 4: Difference maps between images at different concentrations of ATP indicating spread of ATP-induced conformational changes.

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Change history

  • 13 February 2005

    Sentences changed in Methods section

Notes

  1. *Note: In the version of this article originally published online, a mistake was introduced during the production process. This mistake occurs in the last paragraph before the Methods section. The correct first four sentences in this paragraph should read “Our results indicate two important differences between CCT and GroEL that probably have functional importance. First, considerable apparent conformational heterogeneity is seen in the apo state of CCT, but not in GroEL. This heterogeneity decreases with increasing ATP concentration and may reflect protein substrate specificity of different CCT subunits in the protein substrate acceptor state of CCT. Second, our results indicate that the ATP-induced conformational changes in CCT take place in a sequential manner according to the KNF model20, in contrast with the concerted mechanism observed in the case of GroEL.” This error has been corrected for the HTML and print versions of the article. We apologize for any inconvenience this may have caused.

References

  1. Llorca, O. et al. 3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin. Nat. Struct. Biol. 6, 639–642 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Horovitz, A., Fridmann, Y., Kafri, G. & Yifrach, O. Review: Allostery in chaperonins. J. Struct. Biol. 135, 104–114 (2001).

    Article  CAS  Google Scholar 

  4. Saibil, H.R., Horwich, A.L. & Fenton, W.A. Allostery and protein substrate conformational change during GroEL/GroES-mediated protein folding. Adv. Protein Chem. 59, 45–72 (2002).

    Article  CAS  Google Scholar 

  5. Bukau, B. & Horwich, A.L. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351–366 (1998).

    Article  CAS  Google Scholar 

  6. Thirumalai, D. & Lorimer, G.H. Chaperonin-mediated protein folding. Annu. Rev. Biophys. Biomol. Struct. 30, 245–269 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Gutsche, I., Essen, L.O. & Baumeister W. Group II chaperonins: new TRiC(k)s and turns of a protein folding machine. J. Mol. Biol. 293, 295–312 (1999).

    Article  CAS  Google Scholar 

  9. Valpuesta, J.M., Martin-Benito, J., Gomez-Puertas, P., Carrascosa, J.L. & Willison, K.R. Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT. FEBS Lett. 529, 11–16 (2002).

    Article  CAS  Google Scholar 

  10. Spiess, C., Meyer, A.S., Reissmann, S. & Frydman, J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol. 14, 598–604 (2004).

    Article  CAS  Google Scholar 

  11. Liou, A.K.F. & Willison, K.R. Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes. EMBO J. 16, 4311–4316 (1997).

    Article  CAS  Google Scholar 

  12. Llorca, O. et al. Eukaryotic type II chaperonin CCT interacts with actin through specific subunits. Nature 402, 693–696 (1999).

    Article  CAS  Google Scholar 

  13. 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 

  14. 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 

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

    Article  CAS  Google Scholar 

  16. Yifrach, O. & Horovitz, A. Allosteric control by ATP of non-folded protein binding to GroEL. J. Mol. Biol. 255, 356–361 (1996).

    Article  CAS  Google Scholar 

  17. Horovitz, A. & Yifrach, O. On the relationship between the Hill coefficients for steady-state and transient kinetic data: a criterion for concerted transitions in allosteric proteins. Bull. Math. Biol. 62, 241–246 (2000).

    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. Monod, J., Wyman, J. & Changeux, J.-P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118 (1965).

    Article  CAS  Google Scholar 

  20. Koshland, D.E. Jr., Né methy, G. & Filmer, D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5, 365–385 (1966).

    Article  CAS  Google Scholar 

  21. Kafri, G., Willison, K.R. & Horovitz, A. Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1. Protein Sci. 10, 445–449 (2001).

    Article  CAS  Google Scholar 

  22. 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 

  23. Melki, R., Batelier, G., Soulié, S. & Williams, R.C. Jr. Cytoplasmic chaperonin containing TCP-1: structural and functional characterization. Biochemistry 36, 5817–5826 (1997).

    Article  CAS  Google Scholar 

  24. Lin, P. & Sherman, F. The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex. Proc. Natl. Acad. Sci. USA 94, 10780–10785 (1997).

    Article  CAS  Google Scholar 

  25. Hynes, G., Kubota, H. & Willison, K.R. Antibody characterisation of two distinct conformations of the chaperonin-containing TCP-1 from mouse testis. FEBS Lett. 358, 129–132 (1995).

    Article  CAS  Google Scholar 

  26. Frank, J. Three-dimensional Electron Microscopy of Macromolecular Assemblies (Academic Press, San Diego, 1996).

    Google Scholar 

  27. Llorca, O. et al. Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations. EMBO J. 19, 5971–5979 (2000).

    Article  CAS  Google Scholar 

  28. Danziger, O., Rivenzon-Segal, D., Wolf, S.G. & Horovitz, A. Conversion of the allosteric transition of GroEL from concerted to sequential by the single mutation Asp-155→Ala. Proc. Natl. Acad. Sci. USA 100, 13797–13802 (2003).

    Article  CAS  Google Scholar 

  29. Llorca, O. et al. ATP binding induces large conformational changes in the apical and equatorial domains of the eukaryotic chaperonin containing TCP-1 complex. J. Biol. Chem. 273, 10091–10094 (1998).

    Article  CAS  Google Scholar 

  30. Meyer, A.S. et al. Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis. Cell 113, 369–381 (2003).

    Article  CAS  Google Scholar 

  31. Llorca, O. et al. The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin. EMBO J. 20, 4065–4075 (2001).

    Article  CAS  Google Scholar 

  32. Hynes, G.M. & Willison, K.R. Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of β-actin. J. Biol. Chem. 275, 18985–18994 (2000).

    Article  CAS  Google Scholar 

  33. Netzer, W.J. & Hartl, F.U. Recombination of protein domains facilitated by co-translational folding in eukaryotes. Nature 388, 343–349 (1997).

    Article  CAS  Google Scholar 

  34. Ludtke, S.J., Jakana, J., Song, J.-L., Chuang, D.T. & Chiu, W. A 11.5 Å single particle reconstruction of GroEL using EMAN. J. Mol. Biol. 314, 253–262 (2001).

    Article  CAS  Google Scholar 

  35. Frank, J., Shimkim, B. & Dowse, H. SPIDER: a modular software system for electron image processing. Ultramicroscopy 6, 343–358 (1981).

    Article  Google Scholar 

  36. Davies, D.L. & Bouldin, D.W. A cluster separation measure. IEEE Trans. Pattern Anal. Machine Intelligence 1, 224–227 (1979).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by The Israel Science Foundation. D.R.-S is a recipient of a Phil Zacharia postdoctoral fellowship. A.H. is an incumbent of the Carl and Dorothy Bennett Professorial Chair in Biochemistry. K.R.W. is supported by Cancer Research UK. We thank the Institute of Cancer Research Hybridoma Unit for monoclonal antibody production and A. Friesem and A. Pe'er for useful discussion.

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Authors and Affiliations

  1. Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel

    Dalia Rivenzon-Segal, Liat Shimon & Amnon Horovitz

  2. Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, 76100, Israel

    Sharon G Wolf

  3. Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, SW3 6JB, UK

    Keith R Willison

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Correspondence to Amnon Horovitz.

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Rivenzon-Segal, D., Wolf, S., Shimon, L. et al. Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis. Nat Struct Mol Biol 12, 233–237 (2005). https://doi.org/10.1038/nsmb901

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