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Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine

Nature Chemical Biology volume 11, pages 201–206 (2015)Cite this article

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An Erratum to this article was published on 18 March 2015

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Abstract

Hexameric ATP-dependent proteases and protein remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. Whether loops from multiple subunits act independently or coordinately in these processes is a critical aspect of the mechanism but is currently unknown for any AAA+ machine. By studying covalently linked hexamers of the Escherichia coli ClpX unfoldase bearing different numbers and configurations of wild-type and mutant pore loops, we show that loops function synergistically, and the number of wild-type loops required for efficient degradation is dependent on the stability of the protein substrate. Our results support a mechanism in which a power stroke initiated in one subunit of the ClpX hexamer results in the concurrent movement of all six pore loops, which coordinately grip and apply force to the substrate.

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Figure 1: Conserved loops in the axial pore of ClpX and related AAA+ machines mediate translocation and unfolding of polypeptide substrates.
Figure 2: Pore-loop mutants.
Figure 3: Degradation of unfolded UFtitinI27-ssrA.
Figure 4: Unfolding and degradation of native GFP substrates by pore-loop variants of ClpXP.
Figure 5: Maximal rates of GFP-ssrA degradation by ClpXP variants containing one pore-loop mutation (A) and/or one ATP-hydrolysis mutation (EQ) in the same or different subunits supports a model in which the function of a pore loop in one subunit is independent of ATP hydrolysis in the same subunit.
Figure 6: Initiation of GFP-ssrA unfolding.

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  • 30 January 2015

    There was a mismatch between the PDF and the HTML versions upon online release. In addition, the title of the article and the third sentence of the article have been modified by changing 'an' to 'a' before AAA+. The first sentence of the first paragraph was edited incorrectly and has been modified to reflect the correct meaning. Finally, in the last sentence on page 5, the word 'given' has been modified to 'based on'. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by US National Institutes of Health grant GM-101988 (R.T.S.). T.A.B. is an employee of the Howard Hughes Medical Institute. We thank K. Schmitz and E. Gur for materials, advice and helpful discussions.

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Authors and Affiliations

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

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

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

    Tania A Baker

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  1. Ohad Iosefson
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  2. Andrew R Nager
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  3. Tania A Baker
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  4. Robert T Sauer
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Contributions

O.I. and A.R.N. designed and performed experiments. All authors analyzed data and contributed to writing the manuscript.

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Correspondence to Robert T Sauer.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1 and 2, and Supplementary Figures 1–4. (PDF 1651 kb)

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Iosefson, O., Nager, A., Baker, T. et al. Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine. Nat Chem Biol 11, 201–206 (2015). https://doi.org/10.1038/nchembio.1732

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