The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3^KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3^KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3^KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3^KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

CUL3-RING E3 泛素连接酶 (CRL3) 在响应细胞外营养和应激刺激方面发挥着重要作用。 CRL3 的泛素连接酶功能通过二聚化被激活。然而，其连接酶活性如何以及为何需要这样的二聚体组装仍然难以捉摸。在这里，我们报告了二聚 CRL3 ^KLHL22复合物的冷冻电镜结构，并揭示了 CUL3 中的保守 N 末端基序，该基序有助于 CRL3 ^KLHL22的二聚化组装和 E3 连接酶活性。我们发现 CUL3 N 端基序的删除会损害 CRL3 ^KLHL22和其他几个 CRL3 的二聚体组装和 E3 连接酶活性。此外，我们发现 CRL3 ^KLHL22二聚体组装的动力学会产生可变的泛素化区域，可能促进底物识别和泛素化。这些发现表明 CUL3 N 末端基序参与组装过程，并为 CRL3 的组装和激活提供了见解。