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

Alireza Ghanbarpour, Steven E. Cohen, Xue Fei, Laurel F. Kinman, Tristan A. Bell, Jia Jia Zhang, Tania A. Baker, Joseph H. Davis () and Robert T. Sauer ()
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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

Nature Communications, 2023, vol. 14, issue 1, 1-10

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-43145-x

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