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Abstract ClpXP and other AAA proteases maintain proteostasis and regulate cellular functions by degrading misfolded, incomplete, or regulatory proteins. ClpX recognizes substrates via unstructured degron sequences, typically located at the N‐ or C‐terminus. Although five classes of degrons are known, only recognition of the ssrA tag, a C‐motif‐1 degron, is well understood. The ssrA tag initially binds to a conformation of hexameric ClpX in which the axial channel is closed by a pore‐2 loop, with subsequent channel opening allowing translocation into and degradation by ClpP. A ClpX variant with a pore‐2 loop deletion (ΔNPS) favors the open conformation and exhibits weaker binding to ssrA‐tagged substrates. Here, using model substrates representing each of the five known degron classes, we show that ΔNPS ClpXP degrades low micromolar concentrations of N‐motif‐1, ‐2, ‐3, and C‐motif‐2 substrates more effectively than wild‐type ClpXP. Cryo‐EM analysis of wild‐type ClpXP bound to an N‐motif‐1 substrate reveals degron engagement within an open axial channel. Our results support a model in which the open‐ and closed‐channel conformations of ClpXP differentially enhance recognition of distinct degron classes: the open channel facilitates degradation of many natural substrates, whereas the closed channel promotes efficient recognition and degradation of ssrA‐tagged proteins. We propose that the conformational equilibrium between these two states tunes ClpXP activity to balance broad substrate recognition with specificity, allowing cells to meet dynamic proteolytic demands and minimize off‐target degradation.
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A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation
Abstract AAA+ proteases degrade intracellular proteins in a highly specific manner.E. coliClpXP, 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 inE. 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.
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- Award ID(s):
- 2046778
- PAR ID:
- 10493412
- Publisher / Repository:
- Springer Nature
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1038/s41467-023-43145-x
