Abstract
How biological machines harness ATP to drive mechanical work remains a crucial question. Structural studies of protein-translocating AAA+ machines proposed a coupled and sequential translocation process, whereby ATP hydrolysis events lead to short threading steps. Yet, direct real-time observation of these events remains elusive. Here, we employ single-molecule FRET spectroscopy to track substrate translocation through ClpB, a quality control AAA+ machine. We isolate ClpB and its substrate within lipid vesicles and find that translocation events, while dependent on ATP, take milliseconds, much faster than ATP hydrolysis times. Surprisingly, the translocation rate depends weakly on temperature and ATP concentration. Using three-color FRET experiments, we find that translocation events can occur bidirectionally but are not always complete. Replacing ATP with the slowly hydrolysable analog ATPγS abolishes both rapid translocation and directionality. These results indicate a fast, stochastic Brownian-motor-like mechanism, redefining how ATP is coupled with mechanical action in AAA+ machines.
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Data availability
All data generated and processed in this study have been deposited in the Zenodo database under accession code 17532905. A summary of the data in this manuscript is provided in the Supplementary Materials. Source data are provided with this paper. This study used the following PDB structures of ClpB: 6OAX and 1QVR. Source data are provided with this paper.
Code availability
All code generated in this study has been deposited in the Zenodo database under accession code 17532905.
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Acknowledgements
We thank Drs. Pierre Goloubinoff, Hagen Hofmann, Axel Mogk, David Scheerer, Ilya Kuprov, and Michal Haran for reading and commenting on the manuscript. We thank Drs. Hagen Hofmann and Tanya Lasitza-Male for the use of their homebuilt Peltier control unit, Dr. David Scheerer for the fluorescently labeled enzyme adenylate kinase, and Dr. Hisham Mazal for his insightful input and kindly providing samples for initial experimental trials. This work was supported by a grant from the NSF-BSF program (no. 2021700, R.C., D.L., I.R., B.Y., G.H.). R.C. is grateful to the Azrieli Foundation for its generous funding of an Azrieli International Postdoctoral Fellowship.
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R.C., I.R., and G.H. conceptualized and designed the experiments. R.C. conducted the experiments. R.C., D.L., I.R., and Y.B. worked on protein expression, purification, and labeling. R.C. and G.H. analyzed the results and wrote the manuscript. All authors contributed to editing and discussions.
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Casier, R., Levy, D., Riven, I. et al. A stochastic mechanism drives fast substrate translocation in the AAA+ machine ClpB. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68478-1
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DOI: https://doi.org/10.1038/s41467-026-68478-1
