Title:Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination

Brief: Site-selective nucleosome ubiquitination plays critical roles in the epigenetic regulation of multiple chromatin processes. However, elucidating the molecular mechanism of nucleosome ubiquitination is challenging because of the difficulty in capturing the fleeting quaternary complexes of the nucleosome, ubiquitin (Ub), the Ub-conjugating enzyme E2, and the Ub ligase E3. Here, we developed a E2-Ub-nucleosome conjugation strategy in which the Ub C terminus was covalently linked to both the E2 active site and a specific nucleosome lysine residue, imitating the intermediate of Ub transfer from E2 to nucleosome. This E2-Ub-nucleosome conjugation strategy enables, for the first time, constitution of a stable quaternary E3/E2-Ub complex with the nucleosome and determination of its cryo-EM structure at near-atomic resolution. Our work also supports the use of a chemical trapping strategy to study the E3-mediated Ub-transferring mechanisms on full-length and folded proteins.