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A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

Author

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  • Alireza Ghanbarpour

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Steven E. Cohen

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Xue Fei

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Laurel F. Kinman

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Tristan A. Bell

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Jia Jia Zhang

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Tania A. Baker

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Joseph H. Davis

    (Department of Biology Massachusetts Institute of Technology Cambridge)

  • Robert T. Sauer

    (Department of Biology Massachusetts Institute of Technology Cambridge)

Abstract

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.

Suggested Citation

  • Alireza Ghanbarpour & Steven E. Cohen & Xue Fei & Laurel F. Kinman & Tristan A. Bell & Jia Jia Zhang & Tania A. Baker & Joseph H. Davis & Robert T. Sauer, 2023. "A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-43145-x
    DOI: 10.1038/s41467-023-43145-x
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    References listed on IDEAS

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    1. Shuwen Zhang & Shitao Zou & Deyao Yin & Lihong Zhao & Daniel Finley & Zhaolong Wu & Youdong Mao, 2022. "USP14-regulated allostery of the human proteasome by time-resolved cryo-EM," Nature, Nature, vol. 605(7910), pages 567-574, May.
    2. Andreas Martin & Tania A. Baker & Robert T. Sauer, 2005. "Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines," Nature, Nature, vol. 437(7062), pages 1115-1120, October.
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    Cited by:

    1. Alireza Ghanbarpour & Robert T. Sauer & Joseph H. Davis, 2024. "A proteolytic AAA+ machine poised to unfold protein substrates," Nature Communications, Nature, vol. 15(1), pages 1-10, December.

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