Chemical Synthesis of Activity‐Based E2‐Ubiquitin Probes for the Structural Analysis of E3 Ligase‐Catalyzed Transthiolation
Lujun Liang, Guo‐Chao Chu, Qian Qu, Chong Zuo, Junxiong Mao, Qingyun Zheng, Jingnan Chen, Xianbin Meng, Yangwode Jing, Haiteng Deng, Yi‐Ming Li, Lei Liu
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.