MS/MS analysis of Ub-conjugated UbcH7 ubiquitination sites

By conducting the ubiquitin-conjugation assay for UbcH7, we found that PknG was able to promote the loading of Ub onto UbcH7, which is indicated by a molecular weight shift in SDS–PAGE. The classic Ub conjugation of E2s requires the E1 to form a thiol-ester bond between its active Cys and the C-terminal Gly of Ub. Notably, PknG could directly catalyze Ub conjugation onto UbcH7 in a kinase activity-independent and Ubl domain-dependent manner.