Ufmylation is a newly identified ubiquitin-like modification of histones and plays important roles in DNA-related processes. Dissecting histone ufmylation pathways necessitates the use of chemically defined proteins to assign their structural and functional consequences; however, the preparation of ufmylated histones has not yet been reported. Here, we report the chemical synthesis of ufmylated histones and their analogs through semisynthetic strategies integrating chemoenzymatic C-terminal hydrazinolysis of ubiquitin-fold modifier 1 (UFM1) and auxiliary-mediated formation of an isopeptide bond. The results indicated that the E1-mediated activation of UFM1 can be hijacked by nucleophilic reagents, forming the full-length UFM1 hydrazide that can be readily installed onto histones via auxiliary-mediated ligations. The synthetic histones enabled us to reveal that the two known UFM1-specific proteases 1 and 2 (UfSP1 and UfSP2) cannot efficiently cleave H4 ufmylation at Lys31 (H4K31UFM1) in the nucleosome context. Furthermore, cryo-electron microscopy (cryo-EM) analysis of the H4K31UFM1-nucleosome suggested that the steric hindrance of the nucleosome around the isopeptide bond might be one of the potential reasons for the weak activities of UfSPs. Collectively, we developed practical strategies for the efficient generation of ufmylated histones and exemplified their use in biochemical and structural studies related to histone ufmylation.
Liu et al. (Sat,) studied this question.