An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting.

一种新兴的泛素化机制涉及两种不同的 E3 连接酶的合作。在最典型的 E3-E3 通路中，ARIH 家族 RING-between-RING (RBR) E3s 将泛素连接到 neddylated cullin-RING E3s 的底物上。E3 ARIH2 与基于 neddylated CUL5-RBX2 的 E3 底物的泛素化有关，包括被 HIV-1 病毒感染因子 (Vif) 劫持的宿主 E3 的 APOBEC3 家族底物。然而，结构机制仍然难以捉摸。这里的结构和生化分析揭示了独特的 ARIH2 自抑制，以及在与 neddylated CUL5-RBX2 组装时的激活。与包含 ARIH1 和基于 CUL1-RBX1 的 E3 的 E3-E3 组件结构的比较显示了 NEDD8 对 cullin 的特定调节。CUL1 相关的 NEDD8 直接招募 ARIH1，而 CUL5 相关的 NEDD8 不结合 ARIH2。相反，数据揭示了一种变构机制。NEDD8 独特地接触共价连接的 CUL5，并引发结构重排，揭示神秘的 ARIH2 结合位点。数据揭示了泛素样蛋白如何通过变构间接诱导蛋白质-蛋白质相互作用。泛素样蛋白修饰的变构特异性可能为治疗靶向提供机会。