Mechanism-based small molecule cross-linkers of HECT E3 ubiquitin ligase-substrate pairs.

Sungjin Park, Ioanna Ntai, Paul Thomas, Evgeniia Konishcheva, Neil L Kelleher, Alexander V Statsuk

Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Proteomics Center of Excellence, Chemistry of Life Processes Institute, Northwestern University, Silverman Hall, 2145 Sheridan Road, Evanston, IL 60208, USA.

Biochemistry 2012 Oct 23

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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.

Citation

Sungjin Park, Ioanna Ntai, Paul Thomas, Evgeniia Konishcheva, Neil L Kelleher, Alexander V Statsuk. Mechanism-based small molecule cross-linkers of HECT E3 ubiquitin ligase-substrate pairs. Biochemistry. 2012 Oct 23;51(42):8327-9