Hsp100 polypeptide translocases are conserved members of the AAA+ family (adenosine triphosphatases associated with diverse cellular activities) that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from Saccharomyces cerevisiae solubilizes stress-induced amorphous aggregates and amyloids. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo–electron microscopy structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80-angstrom-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 nucleotide hydrolysis sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and substrate-responsive flexibility during Hsp104-catalyzed disaggregation.

Hsp100多肽转座酶是AAA +家族（与多种细胞活性相关的腺苷三磷酸酶）的保守成员，可通过展开异常和有毒的蛋白质进行重新折叠或蛋白水解降解来维持蛋白稳定。酿酒酵母中的Hsp104脱氢酶溶解应力诱导的无定形聚集体和淀粉样蛋白。底物识别和转运的结构基础是未知的。使用模型底物（酪蛋白），我们报道了在不同易位状态下Hsp104在近原子分辨率下的冷冻电子显微镜结构。底物相互作用是由保守的孔环酪氨酸介导的，这些酪氨酸沿轴向通道与80埃长的未折叠多肽接触。两个启动子经历了类似棘轮状的构象变化，使孔环-底物之间的相互作用增加了两个氨基酸。这些变化与特定核苷酸水解位点的激活有关，并且当在六聚体周围传递时，在Hsp104催化的分解过程中显示了一种连续的旋转易位机制和底物响应的灵活性。