Cross-link MS analysis of human 26S proteasome in complex with RPN13:UCHL5 (without substrate).

This project investigates how the human 26S proteasome recognizes and processes proteins tagged with K11/K48-branched ubiquitin chains, which signal for rapid degradation during cell cycle progression and proteotoxic stress. Using cryo-electron microscopy (cryo-EM), the study uncovers a novel K11-linked ubiquitin binding site at the interface of RPN2 and RPN10, alongside known K48-specific sites. Structural insights reveal how these branched ubiquitin chains are preferentially recognized, enhancing proteasomal degradation efficiency. Complementary cross-linking mass spectrometry (XL-MS) identifies transient interactions between RPN13, RPN2, RPN10, and core proteasome subunits, further elucidating the dynamic coordination within the proteasome during substrate processing.