Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
Structure. 2022 Sep 1;30(9):1269-1284.e6. doi: 10.1016/j.str.2022.05.017. Epub 2022 Jun 17.
RING-between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3-ubiquitin thioester intermediate prior to substrate ubiquitination. Although RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic, and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using a combination of XL-MS, HDX-MS, NMR, and biochemical studies. The results show that (1) a di-aromatic surface on the catalytic HHARI Rcat domain forms a binding platform for substrates and (2) a phosphomimetic mutation on the auto-inhibitory Ariadne domain of HHARI promotes release and reorientation of Rcat for transthiolation and substrate modification. The findings identify a direct binding interaction between a RING-between-RING ligase and its substrate and suggest a general model for RBR substrate recognition.
RING 之间的环（RBR）E3 连接酶通过在底物泛素化之前必需的 E3-泛素硫酯中间产物介导泛素转移。尽管 RBR 共享保守的催化模块，但底物募集机制仍然是个谜，并且尚未为该类别的任何成员鉴定相关结构域。在这里，我们使用 XL-MS、HDX-MS、NMR 和生化研究相结合的方法，描述了自动抑制性 RBR HHARI（AriH1）与其靶蛋白 4EHP 之间的相互作用。结果表明：（1）催化 HHARI Rcat 结构域上的双芳香表面形成了结合平台，用于与底物结合；（2）HHARI 自动抑制性 Ariadne 结构域上的磷酸模拟突变促进了 Rcat 的释放和重定向，以进行硫酯转移和底物修饰。这些发现确定了 RING 之间的环连接酶与其底物之间的直接结合相互作用，并提出了 RBR 底物识别的一般模型。
