Most cullin-RING ubiquitin ligases (CRLs) form homologous assemblies between a neddylated cullin-RING catalytic module and a variable substrate-binding receptor (for example, an F-box protein). However, the vertebrate-specific CRL7^FBXW8 is of interest because it eludes existing models, yet its constituent cullin CUL7 and F-box protein FBXW8 are essential for development, and CUL7 mutations cause 3M syndrome. In this study, cryo-EM and biochemical analyses reveal the CRL7^FBXW8 assembly. CUL7’s exclusivity for FBXW8 among all F-box proteins is explained by its unique F-box-independent binding mode. In CRL7^FBXW8, the RBX1 (also known as ROC1) RING domain is constrained in an orientation incompatible with binding E2~NEDD8 or E2~ubiquitin intermediates. Accordingly, purified recombinant CRL7^FBXW8 lacks auto-neddylation and ubiquitination activities. Instead, our data indicate that CRL7 serves as a substrate receptor linked via SKP1–FBXW8 to a neddylated CUL1–RBX1 catalytic module mediating ubiquitination. The structure reveals a distinctive CRL–CRL partnership, and provides a framework for understanding CUL7 assemblies safeguarding human health.

大多数 cullin-RING 泛素连接酶 (CRL) 在 neddylated cullin-RING 催化模块和可变底物结合受体（例如 F-box 蛋白）之间形成同源组装。然而，脊椎动物特异性 CRL7 ^FBXW8很有趣，因为它避开了现有模型，但其成分库林 CUL7 和 F-box 蛋白 FBXW8 对发育至关重要，而 CUL7 突变会导致 3M 综合征。在这项研究中，低温电子显微镜和生化分析揭示了 CRL7 ^FBXW8组装。CUL7 在所有 F-box 蛋白中对 FBXW8 的排他性由其独特的 F-box 独立结合模式解释。在 CRL7 ^FBXW8，RBX1（也称为 ROC1）RING 域被限制在与结合 E2~NEDD8 或 E2~泛素中间体不相容的方向。因此，纯化的重组 CRL7 ^FBXW8缺乏自身内嵌化和泛素化活性。相反，我们的数据表明 CRL7 作为底物受体通过 SKP1–FBXW8 连接到介导泛素化的 neddylated CUL1–RBX1 催化模块。该结构揭示了一种独特的 CRL-CRL 伙伴关系，并提供了一个框架来理解保护人类健康的 CUL7 组件。