Essential Role for Ubiquitin-Ubiquitin-Conjugating Enzyme Interaction in Ubiquitin Discharge from Cdc34 to Substrate

SummaryDuring ubiquitin conjugation, the thioester bond that links “donor” ubiquitin to ubiquitin-conjugating enzyme (E2) undergoes nucleophilic attack by the ɛ-amino group of an acceptor lysine, resulting in formation of an isopeptide bond. Models of ubiquitination have envisioned the donor ubiquitin to be a passive participant in this process. However, we show here that the I44A mutation in ubiquitin profoundly inhibits its ability to serve as a donor for ubiquitin chain initiation or elongation, but can be rescued by computationally predicted compensatory mutations in the E2 Cdc34. The donor defect of ubiquitin-I44A can be partially suppressed either by using a low pKa amine (hydroxylamine) as the acceptor or by performing reactions at higher pH, suggesting that the discharge defect arises in part due to inefficient deprotonation of the acceptor lysine. We propose that interaction between Cdc34 and the donor ubiquitin organizes the active site to promote efficient ubiquitination of substrate.

Graphical AbstractFigure optionsDownload high-quality image (128 K)Download as PowerPoint slideHighlights
► Ubiquitin-I44A mutation is defective in thioester discharge from Cdc34 to substrate
► Ub-I44A thioester discharge defect can be rescued by compensatory mutations in Cdc34
► Interaction between donor Ub and Cdc34 is critical for efficient thioester discharge
► Discharge defect of Ub-I44A is due to inefficient deprotonation of acceptor lysine