A novel regulatory mechanism for the CRL4(DCAF1) RING ubiquitin ligase involving oligomerization

E3 ubiquitin ligases catalyze the transfer of ubiquitin (Ub) from E2~Ub to a substrate. Cullin RING E3 ubiquitin ligases (CRLs) are modular multi-subunit complexes that associate with several substrate-receptor subunits. CRL ligase activity is stimulated by the covalent attachment of the ubiquitin-like molecule NEDD8 (neddylation) to the cullin subunit. The COP9 signalosome complex reverses this activation process by the catalytic cleavage of NEDD8, leading to the inactivation of the ligase. CAND1 is the substrate adaptor-receptor exchange factor, which catalyzes the recruitment of new substrate receptors to the cullin subunit to bind substrates.
The CRL4 (Cullin 4-DDB1-Rbx1) family of cullin-RING E3 ubiquitin ligases employs ~20 different substrate receptors, referred to as DCAFs (DDB1 and Cullin-4 Associated Factors). DCAF1 is a central member of the DCAF family, and found to be dysregulated in different types cancers. The activity of CRL4DCAF1 is suppressed when DCAF1 is bound to Merlin, a tumor suppressor protein that acts upstream of the Hippo pathway. CRL4DCAF1 recruits substrates implicated in DNA repair, cell cycle progression, and cell fate regulation. Viruses also hijack the CRL4 system by binding DCAF1, and drive the degradation of cellular proteins that facilitate viral infections.
The regulatory mechanism underlying CRL4DCAF1 activity is currently poorly understood. We have used biochemical and structural approaches to study CRL4DCAF1 in vitro. The 8.2 Å cryo-EM map of CRL4DCAF1 finds the complex in a tetrameric arrangement, where DCAF1 WD40 domain mediates the interaction with the cullin C-terminal domain (CTD) and Rbx1. This architecture renders the RING domain of Rbx1, the catalytic subunit of the ligase, inaccessible to the E2. Upon activation of CRL4DCAF1 by neddylation, the interaction between the cullin CTD and DCAF1 is broken and the complex becomes dimeric and active. This dimeric state is also induced when the viral proteins VPR-UNG2 are bound to CRL4DCAF1, while the presence of Merlin is compatible with the tetrameric auto-inhibited state of the ligase. Moreover, the tetrameric conformation of CRL4DCAF1 seems to protect the ligase from the spontaneous exchange of adaptor-receptor modules in the absence of CAND1. These results suggest a novel mechanism by which the activity of isolated CRL4DCAF1 ligase is regulated.