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Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX

Nature Structural & Molecular Biology volume 22, pages 411–416 (2015)Cite this article

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Abstract

The hexameric AAA+ ring of Escherichia coli ClpX, an ATP-dependent machine for protein unfolding and translocation, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+-ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U↔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+-ring activity.

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Figure 1: ATP binding and subunit conformations in ring hexamers of ClpX.
Figure 2: Physical characterization of parental and BMH enzymes.
Figure 3: ATP hydrolysis.
Figure 4: Subunit-specific nucleotide binding and effects on ATP hydrolysis in BMH U-locked hexamers.
Figure 5: Binding, unfolding and degradation of protein substrate by ClpX variants.
Figure 6: ATP hydrolysis and degradation by ClpX variants containing two NPM- or BMH-modified subunits.

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Acknowledgements

This research was supported by the US National institutes of Health grant GM-101988 (R.T.S.). B.M.S. was supported by a Massachusetts Institute of Technology Poitras predoctoral fellowship. T.A.B. is supported as an employee of the Howard Hughes Medical Institute.

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Authors and Affiliations

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

    Benjamin M Stinson, Vladimir Baytshtok, Karl R Schmitz, 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. Benjamin M Stinson
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  2. Vladimir Baytshtok
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  4. Tania A Baker
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Contributions

B.M.S., V.B. and K.R.S. designed experiments. B.M.S. performed experiments. B.M.S., T.A.B. and R.T.S. analyzed data and wrote the manuscript. All authors approved the final version of the manuscript.

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Correspondence to Robert T Sauer.

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Supplementary Data Set 1

SDS-PAGE gel shown in Figure 1e (PDF 901 kb)

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Stinson, B., Baytshtok, V., Schmitz, K. et al. Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX. Nat Struct Mol Biol 22, 411–416 (2015). https://doi.org/10.1038/nsmb.3012

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