The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
School of Cellular and Molecular Medicine, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
Sci Rep. 2018 Jan 8;8(1):68. doi: 10.1038/s41598-017-18513-5.
RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism.
环环（RBR）泛素连接酶与多种 E2 酶协同作用，并通过 E3-泛素硫酯中间产物发挥作用。RBR 模块由三个结构域组成，即 RING1、IBR 和 RING2，它们共同协作，将 E2Ub 缀合物中的泛素从 RING1 识别的位置转移到 RING2 中的催化半胱氨酸上，最后在多步反应中将其转移到底物上。最近的研究表明，RING1 结构域以开放构象结合 E2Ub 缀合物，抑制泛素转移到赖氨酸残基上，并促进 E3 硫酯中间产物的形成。然而，E2 的性质如何影响泛素转移过程目前尚不清楚。我们在此报告了对 RBR/E2-缀合物识别步骤的详细表征，表明该机制取决于 E2 酶的性质，并在 UbcH5 和 UbcH7 之间存在差异。在 UbcH5Ub 的情况下，与泛素的相互作用对于稳定转移复合物是必需的，而 UbcH7Ub 的识别主要由 E2-RING1 相互作用驱动。此外，我们的分析表明，RBR 本身及其与负载泛素的 E2 的复合物是动态的，其固有灵活性可能是其催化机制的关键方面。
