Virtually all eukaryotic processes are regulated by cullin-RING E3 ligase (CRL)-catalyzed protein ubiquitylation 1 , which is exquisitely controlled by cullin modification with the ubiquitin (UB)-like protein NEDD8 2 – 6 . However, how CRLs catalyze ubiquitylation, and the basis for NEDD8 activation, remain unknown. We report the cryo EM structure of a chemically-trapped complex representing the ubiquitylation intermediate whereby neddylated CRL1 β-TRCP promotes UB transfer from the E2 UBE2D to its recruited substrate phosphorylated IκBα. The structure shows that NEDD8 acts as a nexus binding disparate cullin elements and the RING-activated UB-linked UBE2D. Concomitant local structural remodeling and large-scale CRL domain movements converge to juxtapose the substrate and ubiquitylation active site. The results explain how a distinctive UB-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 for rapid substrate ubiquitylation and fragile to enable ensuing cullin-RING functions.

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies3–7. Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.

UbFluor is a mechanism-based probe that undergoes a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ligase Rsp5. We show that UbFluor can be utilized to conduct high-throughput screens (HTS) of small molecules against HECT ligases.