Conformational switching of the 26S proteasome enables substrate degradation

The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains, and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the first structure of the 26S proteasome from S. cerevisiae during substrate degradation, showing that the regulatory particle switches from a pre-engaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase, and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Importantly, Rpn11 moves from an occluded position to directly above the central pore, facilitating substrate deubiquitination concomitant with translocation.