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Mechanochemical basis of protein degradation by a double-ring AAA+ machine

Nature Structural & Molecular Biology volume 21, pages 871–875 (2014)Cite this article

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Abstract

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.

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Figure 1: Single-molecule protein degradation by ClpAP.
Figure 2: ClpAP unfolding of different protein domains.
Figure 3: Polypeptide translocation.
Figure 4: Degradation of GFP-ssrA.

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute (HHMI) (T.A.B.) and US National Institutes of Health (grant AI-16892; R.T.S.). T.A.B. is supported as an employee of HHMI. We thank T. L. Sherpa for help, S. Calmat, A. Torres-Delgado and E. Vieux (Massachusetts Institute of Technology) for providing proteins and M. Aubin-Tam (Technische Universiteit Delft) and M. Lang (Vanderbilt University) for discussions and advice.

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  1. Andrew R Nager

    Present address: Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA.,

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Adrian O Olivares, Andrew R Nager, Ohad Iosefson, Robert T Sauer & Tania A Baker

  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Tania A Baker

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Contributions

A.O.O. performed and analyzed optical-trapping experiments, performed biochemical experiments and constructed and purified ClpA variants. A.R.N. constructed, purified and assembled the biotinylated, mixed-ring ClpP enzyme used for single-molecule studies. O.I. constructed and purified the multidomain titin-GFP substrate. All authors contributed to writing the manuscript.

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Correspondence to Robert T Sauer or Tania A Baker.

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Olivares, A., Nager, A., Iosefson, O. et al. Mechanochemical basis of protein degradation by a double-ring AAA+ machine. Nat Struct Mol Biol 21, 871–875 (2014). https://doi.org/10.1038/nsmb.2885

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