Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Nature. 2020 Feb;578(7795):461-466. doi: 10.1038/s41586-020-2000-y. Epub 2020 Feb 12.
Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1 promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
真核细胞生物学依赖于 Cullin-RING E3 连接酶（CRL）催化的蛋白质泛素化，该过程受 Cullin 与泛素样蛋白 NEDD8 修饰的严格调控。然而，CRLs 如何催化泛素化以及 NEDD8 激活的基础仍不清楚。在此，我们报告了一种化学捕获复合物的冷冻电镜结构，该复合物代表了泛素化中间产物，其中 neddylated CRL1 促进了泛素从 E2 泛素连接酶 UBE2D 向其募集的底物磷酸化 IκBα的转移。NEDD8 作为一个连接点，将不同的 Cullin 元件和被 RING 激活的连接泛素的 UBE2D 结合在一起。NEDD8 的局部结构重塑和 CRL 结构域的大规模运动汇聚在一起，使底物和泛素化活性位点并列。这些发现解释了一种独特的泛素样蛋白如何改变其靶标的功能，并展示了许多 NEDD8 依赖性的蛋白间相互作用和构象变化如何协同配置一个催化 CRL 结构，该结构既坚固，能够快速泛素化底物，又脆弱，能够使 Cullin-RING 蛋白的后续功能发挥。
