CULLIN-RING ligases constitute the largest group of E3 ubiquitin ligases. While some CULLIN family members recruit adapters before engaging further with different substrate receptors, homo-dimeric BTB-Kelch family proteins combine adapter and substrate receptor into a single polypeptide for the CULLIN3 family. However, the entire structural assembly and molecular details have not been elucidated to date. Here, we present a cryo-EM structure of the CULLIN3^RBX1 in complex with Kelch-like protein 22 (KLHL22) and a mitochondrial glutamate dehydrogenase complex I (GDH1) at 3.06 Å resolution. The structure adopts a W-shaped architecture formed by E3 ligase dimers. Three CULLIN3^{KLHL22−RBX1} dimers were found to be dynamically associated with a single GDH1 hexamer. CULLIN3^{KLHL22−RBX1} ligase mediated the polyubiquitination of GDH1 in vitro. Together, these results enabled the establishment of a structural model for understanding the complete assembly of BTB-Kelch proteins with CULLIN3 and how together they recognize oligomeric substrates and target them for ubiquitination.

CULLIN-RING 连接酶构成最大的 E3 泛素连接酶组。虽然一些 CULLIN 家族成员在进一步与不同的底物受体结合之前招募衔接子，但同源二聚体 BTB-Kelch 家族蛋白将衔接子和底物受体组合成 CULLIN3 家族的单个多肽。然而，迄今为止，整个结构组装和分子细节尚未阐明。在这里，我们展示了 CULLIN3 ^RBX1与 Kelch 样蛋白 22 (KLHL22) 和线粒体谷氨酸脱氢酶复合物 I (GDH1) 复合物的冷冻电镜结构，分辨率为 3.06 Å。该结构采用E3连接酶二聚体形成的W形结构。发现三个 CULLIN3 ^{KLHL22−RBX1}二聚体与单个 GDH1 六聚体动态相关。 CULLIN3 ^{KLHL22−RBX1}连接酶在体外介导 GDH1 的多泛素化。总之，这些结果使得能够建立一个结构模型，用于了解 BTB-Kelch 蛋白与 CULLIN3 的完整组装，以及它们如何一起识别寡聚底物并将其靶向泛素化。