In the Escherichia coli ClpXP protease, a hexameric ClpX ring couples ATP binding and hydrolysis to mechanical protein unfolding and translocation into the ClpP degradation chamber. Rigid-body packing between the small AAA+ domain of each ClpX subunit and the large AAA+ domain of its neighbor stabilizes the hexamer. By connecting the parts of each rigid-body unit with disulfide bonds or linkers, we created covalently closed rings that retained robust activity. A single-residue insertion in the hinge that connects the large and small AAA+ domains and forms part of the nucleotide-binding site uncoupled ATP hydrolysis from productive unfolding. We propose that ATP hydrolysis drives changes in the conformation of one hinge and its flanking domains and that the changes are propagated around the AAA+ ring through the topologically constrained set of rigid-body units and hinges to produce coupled ring motions that power substrate unfolding.
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This work was supported by US National Institutes of Health grant AI-15706 (R.T.S.). T.A.B. is an employee of the Howard Hughes Medical Institute. We thank B. Stinson and B. Sosa for help and discussions and P. Schwille (Dresden University of Technology) for providing the Kaede plasmid.
The authors declare no competing financial interests.
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Glynn, S., Nager, A., Baker, T. et al. Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine. Nat Struct Mol Biol 19, 616–622 (2012). https://doi.org/10.1038/nsmb.2288
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