Abstract
Proteolytic AAA+ unfoldases use ATP hydrolysis to power conformational changes that mechanically denature protein substrates and then translocate the polypeptide through a narrow pore into a degradation chamber. We show that a tyrosine residue in a pore loop of the hexameric ClpX unfoldase links ATP hydrolysis to mechanical work by gripping substrates during unfolding and translocation. Removal of the aromatic ring in even a few ClpX subunits results in slippage, frequent failure to denature the substrate and an enormous increase in the energetic cost of substrate unfolding. The tyrosine residue is part of a conserved aromatic-hydrophobic motif, and the effects of mutations in both residues vary with the nucleotide state of the resident subunit. These results support a model in which nucleotide-dependent conformational changes in these pore loops drive substrate translocation and unfolding, with the aromatic ring transmitting force to the polypeptide substrate.
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Acknowledgements
This research was supported by the US National Institutes of Health grant AI-15706. We thank J. Davis, E. Gur, A. Keating, M. Laub and S. Sundar for helpful discussions. A.M. was supported by a Merck/Massachusetts Institute of Technology CSBi postdoctoral fellowship. T.A.B. is an employee of the Howard Hughes Medical Institute.
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A.M. designed and performed experiments; A.M., T.A.B. and R.T.S. analyzed data and wrote the manuscript.
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Martin, A., Baker, T. & Sauer, R. Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nat Struct Mol Biol 15, 1147–1151 (2008). https://doi.org/10.1038/nsmb.1503
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DOI: https://doi.org/10.1038/nsmb.1503
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