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.

The human alpha-Synuclein (αS) protein is of significant interest because of its association with Parkinson's disease and related neurodegenerative disorders. The intrinsically disordered protein (140 amino acids) is characterized by the absence of a well-defined structure in solution. It displays remarkable conformational flexibility upon macromolecular interactions, and can associate with mitochondrial membranes. Site-directed spin-labeling in combination with electron paramagnetic resonance spectroscopy enabled us to study the local binding properties of αS on artificial membranes (mimicking the inner and outer mitochondrial membranes), and to evaluate the importance of cardiolipin in this interaction. With pulsed, two-frequency, double-electron electron paramagnetic resonance (DEER) approaches, we examined, to the best of our knowledge for the first time, the conformation of αS bound to isolated mitochondria.

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RBR domains, a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.