The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4^DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4^DCAF1, four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4^DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4^DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4^DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.

中文翻译：

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CRL4DCAF1 cullin-RING 泛素连接酶在寡聚化状态切换后被激活

基于 cullin-4 的环型 (CRL4) E3 泛素连接酶家族与专用底物受体一起发挥作用。在已报道的约 29 个 CRL4 底物受体中，DDB1 和 CUL4 相关因子 1 (DCAF1) 对于细胞存活和生长至关重要，并且其失调与肿瘤发生有关。我们进行了生化和结构研究，以检查 CRL4 ^DCAF1连接酶的结构和机制。在 CRL4 ^DCAF1的 8.4 Å 冷冻电镜图中，四个 CUL4-RBX1-DDB1-DCAF1 原体被组织成两个二聚体子组件。在这种排列中，DCAF1 的 WD40 域介导与 cullin C 端域 (CTD) 和相邻 CRL4 ^DCAF1的 RBX1 亚基的^结合原体。这使得连接酶的催化亚基 RBX1 无法被 E2 泛素结合酶访问。在通过 neddylation 激活CRL4 ^{DCAF1 后}，cullin CTD 与相邻 DCAF1 原体之间的相互作用被破坏，复合物呈现活性二聚体构象。因此，与野生型相比，四聚化缺陷型 CRL4 ^DCAF1突变体具有更高的泛素连接酶活性。本研究确定了一种新机制，通过该机制，未内化和无底物的 CUL4 连接酶可以保持在非活性状态。