Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Centre of Biophotonics, School of Physics and Astronomy, University of St. Andrews, St. Andrews, KY16 9SS, UK.
Nat Commun. 2020 Jun 5;11(1):2846. doi: 10.1038/s41467-020-16666-y.
Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2ubiquitin conjugate (E2Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.
基于广泛的结构分析，人们提出 RING E3 连接酶通过将 E2泛素缀合物（E2Ub）锁定在封闭构象中，从而为催化作用做好准备，在这种构象中，泛素折叠回 E2 上，暴露出受约束的硫酯键，以便底物亲核试剂攻击。然而，RING 依赖性 E2Ub 的封闭构象代表介导泛素转移的活性形式的观点尚未经过实验测试。为了检验这一假设，我们使用单分子Förster 共振能量转移（smFRET）来测量 FRET 标记的 E2Ub 缀合物的构象，该构象可区分封闭和替代构象。我们描述了一种带有硫酯键的 E2~Ub 缀合物的实时 FRET 测定法，以监测单个泛素化事件，并证明泛素从封闭构象转移到底物上。这些发现可能与所有 RING E3 催化反应有关，这些反应将泛素和其他泛素样蛋白（Ubls）连接到底物上。
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