Chemical Synthesis of Activity‐Based E2‐Ubiquitin Probes for the Structural Analysis of E3 Ligase‐Catalyzed Transthiolation. (22nd June 2021)

• Record Type: Journal Article
• Authors: Liang, Lu‐Jun; Chu, Guo‐Chao; Qu, Qian; Zuo, Chong; Mao, Junxiong; Zheng, Qingyun; Chen, Jingnan; Meng, Xianbin; Jing, Yangwode; Deng, Haiteng; Li, Yi‐Ming; Liu, Lei
• Is Part Of: Angewandte Chemie. Volume 133:Number 31(2021)
• Page Start: 17308
• Page End: 17314
• Publication Date: 2021-06-22
• Subjects: chemical protein synthesis -- CXMS -- E2 conjugating enzyme -- probe -- ubiquitin; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.202105870
• Languages: English
• ISSNs: 0044-8249

Abstract

Abstract: Activity‐based E2 conjugating enzyme (E2)‐ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)‐catalyzed ubiquitination. However, the preparation of existing activity‐based E2‐Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide‐based native chemical ligation, which enabled the construction of a structurally new activity‐based E2‐Ub probe to covalently capture the catalytic site of Cys‐dependent E3s. Chemical cross‐linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin‐mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies. Abstract : A structurally new activity‐based E2‐Ub probe that closely resembles the native E2∼Ub thioester has been developed and used for structural analysis of Cys‐dependent E3 ligases‐catalyzed transthiolation. This new probe is readily prepared in an expedient total chemical synthesis of atomically‐tailored E2 enzyme variant using hydrazide‐based native chemical ligation.