RNF213 is the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that often leads to brain stroke in adults and children. Characterization of disease-associated mutations has been complicated by the enormous size of RNF213. Here, we present the cryo-EM structure of mouse RNF213. The structure reveals the intricate fold of the 584 kDa protein, comprising an N-terminal stalk, a dynein-like core with six ATPase units, and a multidomain E3 module. Collaboration with UbcH7, a cysteine-reactive E2, points to an unexplored ubiquitin-transfer mechanism that proceeds in a RING-independent manner. Moreover, we show that pathologic MMD mutations cluster in the composite E3 domain, likely interfering with substrate ubiquitination. In conclusion, the structure of RNF213 uncovers a distinct type of an E3 enzyme, highlighting the growing mechanistic diversity in ubiquitination cascades. Our results also provide the molecular framework for investigating the emerging role of RNF213 in lipid metabolism, hypoxia, and angiogenesis.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure where the C-terminal HECT domain heads an extended alpha solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins. c Ub-charged HECT E3 C2476 catalytic site substrate-binding sites UbcH5b-Ub / NEDD4L(HECT) Rsp5 (HECT)-Ub steric clash: C-lobe HECT : ARM8-10

RNF213 is a giant E3 ubiquitin ligase and a major susceptibility factor of Moyamoya disease, a cerebrovascular disorder that can result in stroke or death. In the cell, RNF213 is involved in lipid droplet formation, lipotoxicity, hypoxia, and NF-κB signaling, but its exact function in these processes is unclear. Structural characterization has revealed the presence of a dynein-like ATPase module and an unprecedented but poorly understood E3 module. Here, we demonstrate that RNF213 E3 activity is dependent on ATP binding, rather than ATP hydrolysis, and is particularly responsive to the ATP/ADP/AMP ratio. Biochemical and activity-based probe analyses identify a non-canonical zinc finger domain as the E3 active site, which utilizes the strictly conserved Cys4462, not involved in zinc coordination, as the reactive nucleophile. The cryo-EM structure of the trapped RNF213:E2~Ub intermediate reveals RNF213 C-terminal domain as the E2 docking site, which positions the ubiquitin-loaded E2 proximal to the catalytic zinc finger, facilitating nucleophilic attack of Cys4462 on the E2~Ub thioester. Our findings show that RNF213 represents an undescribed type of a transthiolation E3 enzyme and is regulated by adenine nucleotide concentration via its ATPase core, possibly allowing it to react to changing metabolic conditions in the cell.