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Distinct structural elements of the adaptor ClpS are required for regulating degradation by ClpAP

Nature Structural & Molecular Biology volume 15, pages 288–294 (2008)Cite this article

Abstract

Adaptor proteins modify substrate recognition by AAA+ ATPases. We examined how the adaptor ClpS regulates substrate choice by the Escherichia coli protease ClpAP. Binding of six ClpS molecules to a ClpA hexamer enhanced N-end-rule substrate degradation and inhibited ssrA-tagged protein proteolysis. Substoichiometric ClpS binding allowed intermediate degradation of both substrate types, revealing that adaptor stoichiometry influences substrate choice. ClpS controls substrate selection using distinct mechanisms. The N-terminal segment is essential for delivering N-end-rule substrates but dispensable for ssrA-protein inhibition. We tested existing models for ClpS action and found that ClpS does not block recognition of ssrA-tagged substrates by steric occlusion and that adaptor-mediated tethering of N-end-rule substrates to ClpAP was insufficient to explain facilitated delivery. We propose that ClpS functions, at least in part, as an allosteric effector of ClpAP, broadening our understanding of how AAA+ adaptors control substrate selection.

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Figure 1: Dual roles of ClpS in changing ClpAP substrate preference.
Figure 2: Stoichiometry of ClpS.
Figure 3: Co-degradation of GFP-ssrA and YLFVQ-titinI27.
Figure 4: ClpS N-terminal extension is not a steric inhibitor of GFP-ssrA.
Figure 5: ClpS N-terminal extension is required for active delivery of YLFVQ-titinI27 and for effect on ClpAP ATPase rate.
Figure 6: Model for ClpS-mediated change in ClpA binding preferences.

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Acknowledgements

We thank K. Wang (Massachusetts Institute of Technology) for generously providing N-end-rule proteins and P. Chien, M. Laub, T. Schwartz, K. Wang, C. Wu and members of the Baker and Sauer Labs for discussion and advice. Plasmid clpA M169T/pET9a was a gift from J. Flanagan (Hershey Medical Center). T.A.B. is an employee of the Howard Hughes Medical Institute. This work was supported by US National Institutes of Health grant GM49224 and the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biology, 68-523, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Jennifer Y Hou, Robert T Sauer & Tania A Baker

  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, 68-523, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Tania A Baker

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  1. Jennifer Y Hou
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  2. Robert T Sauer
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J.Y.H. carried out the experiments. J.Y.H., R.T.S. and T.A.B. contributed to experimental design and wrote the manuscript.

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Correspondence to Tania A Baker.

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Hou, J., Sauer, R. & Baker, T. Distinct structural elements of the adaptor ClpS are required for regulating degradation by ClpAP. Nat Struct Mol Biol 15, 288–294 (2008). https://doi.org/10.1038/nsmb.1392

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