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.

INTRODUCTION: As the major protease in eukaryotic cells and the final component of the ubiquitin-proteasome system, the 26S proteasome is responsible for protein homeostasis and the regulation of numerous vital processes. Misfolded, damaged, or obsolete regulatory proteins are marked for degradation by the attachment of polyubiquitin chains, which bind to ubiquitin receptors of the proteasome. Aheterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) subunits then uses conserved pore loops to engage, mechanically unfold, and translocate protein substrates into a proteolytic core for cleavagewhile the deubiquitinase Rpn11 removes substrateattached ubiquitin chains. RATIONALE: Despite numerous structural and functional studies, the mechanisms by which adenosine triphosphate (ATP) hydrolysis drives the conformational changes responsible for protein degradation remained elusive. Structures of related homohexameric AAA+ motors, in which bound substrates were stabilized with ATP analogs or hydrolysis-eliminating mutations, revealed snapshots of ATPase subunits in different nucleotide states and spiralstaircase arrangements of pore loops around the substrate. These structures gave rise to “handover-hand” translocation models by inferring how individual subunits may progress through various substrate-binding conformations. However, the coordination of ATP-hydrolysis steps and their mechanochemical coupling to propelling substrate were unknown. RESULTS: We present the cryo–electron microscopy (cryo-EM) structures of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome with four distinct motor conformations. Stalling substrate translocation at a defined position by inhibiting deubiquitination led to trapped states in which the substrate-attached ubiquitin remains functionally bound to the Rpn11 deubiquitinase, and the scissile isopeptide bond of ubiquitin is aligned with the substrate-translocation trajectory through the AAA+ motor. Our structures suggest a ubiquitin capture mechanism, in which mechanical pulling on the substrate by the AAA+ motor delivers ubiquitin modifications directly into the Rpn11 catalytic groove and accelerates isopeptide cleavage for efficient, cotranslocational deubiquitination. These structures also show how the substrate polypeptide traverses from the Rpn11 deubiquitinase, through the AAA+ motor, and into the core peptidase. The proteasomal motor thereby adopts staircase arrangements with five substrate-engaged subunits and one disengaged subunit. Four of the substrate-engaged subunits are ATP bound, whereas the subunit at the bottom of the staircase and the disengaged subunit are bound to adenosine diphosphate (ADP). CONCLUSION: Of the four distinct motor states we observed, three apparently represent sequential stages of ATP binding, hydrolysis, and substrate translocation and hence reveal the coordination of individual steps in the ATPase cycle and their mechanochemical coupling with translocation. ATP hydrolysis occurs in the fourth substrate-engag...

ClpB and Hsp104 are conserved AAA+ protein disaggregases that promote survival during cellular stress. Hsp104 acts on amyloids, supporting prion propagation in yeast, and can solubilize toxic oligomers connected to neurodegenerative diseases. A definitive structural mechanism, however, has remained elusive. We have determined the cryo-EM structure of Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the twelve AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains interact to form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, explaining roles in hydrolysis and disaggregation. Remarkably, substrate-binding pore loops line the channel in a continuous spiral that appears optimized for substrate transfer across the AAA+ domains, establishing a directional path for polypeptide translocation.