Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation

作     者：Lu-Jun Liang Guo-Chao Chu Qian Qu Chong Zuo Junxiong Mao Qingyun Zheng Jingnan Chen Xianbin Meng Yangwode Jing Prof. Haiteng Deng Prof. Yi-Ming Li Prof. Lei Liu 

作者机构：Tsinghua-Peking Center for Life Sciences Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology Center for Synthetic and Systems Biology Department of Chemistry Tsinghua University Beijing 100084 China School of Food and Biological Engineering Engineering Research Center of Bio-process Ministry of Education Hefei University of Technology Hefei 230009 China MOE Key Laboratory of Bioinformatics School of Life Sciences Tsinghua University Beijing 100084 China 

出 版 物：《Angewandte Chemie》 (应用化学)

年 卷 期：2021年第133卷第31期

页      面：17308-17314页

学科分类：081704[工学-应用化学] 08[工学] 0817[工学-化学工程与技术] 

主　　题：chemical protein synthesis CXMS E2 conjugating enzyme probe ubiquitin 

摘      要：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.