Structural basis for the ubiquitin chain recognition of the human 26S proteasome

Proteasomal degradation is a fundamental process for all eukaryotic life. A protein destined for degradation is first tagged with a polyubiquitin chain, which is selected by the proteasome. Different ubiquitin chain topologies serve as distinct signals, with K48-linked chains acting as the canonical degradation signal and K11/K48-branched chains providing even more potent targeting, particularly during cell cycle regulation. However, the structural basis for how the proteasome distinguishes between these different chain architectures has remained unclear. Here, we present high-resolution cryo-EM structures of the human 26S proteasome bound to both a K48-linked tetraubiquitin chain and a K11/K48-branched chain. Our structures reveal distinct binding modes for these two types of chain linkage. K48 chains wrap around the Ubiquitin interaction motif of the receptor RPN10 in an unexpected spiral conformation, while K11 branches engage the proteasome through previously uncharacterised interfaces in a cleft formed between RPN2 and RPN10. Through structure-guided mutagenesis and cellular studies, we demonstrate that these binding modes are essential for efficient substrate degradation and cell cycle progression. These findings establish how the proteasome achieves selective substrate recognition through chain topology-specific interactions.