Mechanism-Based Small Molecule Cross-Linkers of HECT E3 Ubiquitin Ligase鈥揝ubstrate Pairs

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• 作者：Sungjin Park ; Ioanna Ntai ; Paul Thomas ; Evgeniia Konishcheva ; Neil L. Kelleher ; Alexander V. Statsuk
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 23, 2012
• 年：2012
• 卷：51
• 期：42
• 页码：8327-8329
• 全文大小：180K
• 年卷期：v.51,no.42(October 23, 2012)
• ISSN：1520-4995

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.