Mechanism-Based Small Molecule Cross-Linkers of HECT E3 Ubiquitin Ligase–Substrate Pairs
2012
Park, Sungjin | Ntai, Ioanna | Thomas, Paul | Konishcheva, Evgeniia | Kelleher, Neil L. | Statsuk, Alexander V.
Here we report the discovery that bifunctional thiol- and amine-reactive electrophiles serve as mechanism-based covalent cross-linkers for HECT E3 ubiquitin ligase–substrate 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–substrate pairs that converts transiently interacting HECT E3–substrate pairs into stable, covalently cross-linked protein complexes, thereby facilitating their subsequent isolation, identification, and study.
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