Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia.
Department of Medical Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia.
Nat Commun. 2023 Jan 11;14(1):168. doi: 10.1038/s41467-023-35871-z.
The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 ubiquitin-conjugating enzyme to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we here structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, together with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric activation mechanism by distinct ubiquitin linkages that suggests RBRs employ a feed-forward mechanism. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 binuclear cluster that is required for catalytic activity and substrate ubiquitination.
人类中的 RING-between-RING (RBR) E3 泛素连接酶家族由 14 个成员组成，其催化机制分为两步，首先泛素从 E2 泛素连接酶转移到 RBR 活性位点，然后转移到底物。为了定义这个催化机制的核心特征，我们在此结构和生化上对两个 RBRs HOIL-1 和 RNF216 进行了表征。捕获第一步催化反应的两种酶的 RBR/E2-Ub/Ub 转硫醇复合物的晶体结构，以及互补的功能实验，揭示了 RBR 催化机制的特征。RBR 通过保守的转硫醇复合物结构催化泛素化，从而实现有效的 E2 到 RBR 泛素转移。我们的数据还突出了不同泛素连接所产生的保守的 RBR 变构激活机制，这表明 RBR 采用了前馈机制。我们最后确定 HOIL-1 的 RING2 结构域包含一个不寻常的 Zn2/Cys6 双核簇，该簇对于催化活性和底物泛素化是必需的。
