Mechanisms of Ub-independent ODC recognition and translocation by the 26S proteasome revealing proteasome's functional adaptability

While most eukaryotic proteins require ubiquitination for proteasomal degradation, a considerable number of substrates undergo ubiquitin-independent degradation. Ornithine decarboxylase (ODC) is a well-known substrate degraded by the 26S proteasome in a ubiquitin-and cofactor-independent manner. However, the underling mechanism of how ODC is recognized and processed without the ubiquitin tag by the 26S proteasome remain unclear. Here, we present eleven cryo-EM structures of the human 26S proteasome in complex with C-terminal truncated or full-length ODC, capturing the entire degradation process from initial recognition to final unfolding. These structures reveal a dynamic, multivalent recognition process involving sequential engagement with vWA domain of Rpn10, Rpt4/5s coiled-coil, and Rpn2s PC domain, collectively bypassing the need for ubiquitin. Although the folded ODC core is sufficient for initial binding, its flexible C-terminal tail is crucial for allosterically activating the proteasomes AAA+ ATPase motor. Furthermore, we also identify a unique translocation gateway where Rpn11s JAMM motif guides the ODC C-tail into the AAA+ATPase ring, repurposing this deubiquitinase for a novel function. Once activated, the proteasome translocates ODC C-tail and unfolds ODC using its canonical ATP-driven machinery. The continuous structural series from initial substrate recognition through final unfolding elucidates the complete molecular mechanism of ubiquitin-independent ODC degradation.