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Global unfolding of a substrate protein by the Hsp100 chaperone ClpA

Nature volume 401, pages 90–93 (1999)Cite this article

Abstract

The bacterial protein ClpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP (refs 1, 2). ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences3,4,5, much as the 19S ATPase ‘cap’ of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins6,7,8. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins9,10; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates11. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder12,13,14. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation5,15. Fluorescence studies both with and without a ‘trap’ version of the chaperonin GroEL, which binds non-native forms of GFP16, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.

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Figure 1: GFP11, a derivative bearing an 11-amino-acid C-terminal ssrA tag recognized by ClpA, exhibits the same stability to denaturants as untagged GFP, and is degraded by ClpA/ClpP in the presence of ATP.
Figure 2: ClpA, in the absence of ClpP, mediates ATP-dependent unfolding of GFP11.
Figure 3: Transfer of GFP11 to GroEL trap in the presence of ClpA and ATP.
Figure 4: Mass spectra of GFP11 subjected to hydrogen–deuterium exchange followed by incubation of the deuterated protein in H2O buffer with or without ClpA and nucleotide.

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Acknowledgements

E.U.W. is a postdoctoral associate of the Jane Coffin Childs Foundation. A.D.M. is a Pew Scholar in the biomedical sciences. This work was supported by the NIH, the Howard Hughes Medical Institute and the Nelson Fund.

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Authors and Affiliations

  1. Department of Genetics,

    Eilika U. Weber-Ban, Brian G. Reid & Arthur L. Horwich

  2. Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center, 295 Congress Avenue, New Haven, 06510, Connecticut, USA

    Brian G. Reid & Arthur L. Horwich

  3. Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, 06520, Connecticut, USA

    Andrew D. Miranker

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Correspondence to Arthur L. Horwich.

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Weber-Ban, E., Reid, B., Miranker, A. et al. Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90–93 (1999). https://doi.org/10.1038/43481

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