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 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 substrate, and an enormous increase in the energetic cost of substrate unfolding. The tyrosine 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, supporting 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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