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ACS Chem Biol. 2016 Jun 17;11(6):1552-1560. doi: 10.1021/acschembio.6b00083. Epub 2016 Mar 30.

Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle.

Author information

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Department of Chemistry, Brown University, Providence, RI 02912.
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
Contributed equally


The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.

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