文摘
Here we report the discovery that bifunctional thiol- and amine-reactive electrophiles serve as mechanism-based covalent cross-linkers for HECT E3 ubiquitin ligase鈥搒ubstrate pairs. We demonstrate that these chemical cross-linkers covalently cross-link the catalytic Cys residue of the yeast HECT E3 ubiquitin ligase Rsp5 with the Lys of the ubiquitination site in the model substrate Sic60-GFP. This work represents the first example of a mechanism-based covalent cross-link of HECT E3鈥搒ubstrate pairs that converts transiently interacting HECT E3鈥搒ubstrate pairs into stable, covalently cross-linked protein complexes, thereby facilitating their subsequent isolation, identification, and study.