Hsp100 polypeptide translocases are conserved AAA+ machines that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from S. cerevisiae solubilizes stress-induced amorphous aggregates and amyloid. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-EM structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80 Å-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore-loop-substrate interactions by two-amino acids. These changes are coupled to activation of specific ATPase sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and a remarkable structural plasticity of Hsp104-catalyzed disaggregation.
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