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Nucleotide-dependent substrate recognition by the AAA+ HslUV protease

Nature Structural & Molecular Biology volume 12, pages 245–251 (2005)Cite this article

Abstract

ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg2+. Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.

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Figure 1: HslUV degradation of Arc repressor and variants.
Figure 2: HslUV degradation of Arc fusion proteins.
Figure 3: Mapping targeting signals for HslUV degradation.
Figure 4: Peptide binding and inhibition.
Figure 5: Nucleotide-dependent binding of the gt1 peptide and Arc to HslU.

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Acknowledgements

We thank J. Flynn and S. Siddiqui for materials and assistance with experiments and members of the Baker and Sauer labs for advice and comments. This work was supported by US National Institutes of Health grants AI-15706 and AI-16892. T.A.B. is an employee of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Randall E Burton, Tania A Baker & Robert T Sauer

  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Tania A Baker

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Correspondence to Robert T Sauer.

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Burton, R., Baker, T. & Sauer, R. Nucleotide-dependent substrate recognition by the AAA+ HslUV protease. Nat Struct Mol Biol 12, 245–251 (2005). https://doi.org/10.1038/nsmb898

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