E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP

Abstract

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.

Moyamoya disease emerging as an immune-related angiopathy

“…The authors suggest that decreased RNF213 E3 ligase activity is central to MMD pathogenesis and they even hypothesize that the extent of impairment might correlate with the level of penetrance of a certain MMD polymorphism. Interestingly, the RNF213 E3 module contains two E3 catalytic domains, a classic RING domain, [54,61] and a noncanonical RZ finger recently described by Otten et al [58] (Figure 2). While the RZ finger is responsible for the ubiquitylation of bacterial lipopolysaccharide (LPS) [58], other (non-)proteinaceous substrates might exist, and further research is needed to elucidate the function and full substrate repertoire of both catalytic domains.…”

The ubiquitin ligation machinery in the defense against bacterial pathogens

“…However, Otten and colleagues mapped a molecular motif within the E3 module, named RZ‐finger, that is typical for zinc‐binding domains, and showed that it is required for LPS ubiquitylation. Recent biochemical studies pinpointed an active site cysteine within the RZ finger that accepts Ub from the E2 enzyme UBCH7, suggesting that RNF213 represents an undescribed type of transthiolation E3 enzyme (preprint: Ahel et al , 2021). RNF213 knockout mouse embryonic fibroblasts (MEFs) or cells expressing RNF213 mutations corresponding to the RZ‐finger are severely defective in Ub coat formation around cytosolic Salmonella and also failed to recruit LUBAC and lacked LUBAC‐mediated M1 Ub chain synthesis.…”

Bacterial lipids earmarked with ubiquitin for pathogen clearance

“…RNF213 is a giant 600 kDa protein with a unique structure that combines a dynein-like ATPase core with a RINGtype E3 ligase domain (Figure 1; Ahel et al, 2020). Interestingly, the RING domain is not required for auto-ubiquitination or for LPS ubiquitination (Otten et al, 2021;Ahel et al, 2020;Ahel et al, 2021). Instead, an adjacent zinc-binding ''RZ'' domain uses a cysteine-dependent mechanism to conjugate ubiquitin, akin to the HECT, RBR, and RCR families of E3 ligases (Otten et al, 2021;Ahel et al, 2021).…”

“…Interestingly, the RING domain is not required for auto-ubiquitination or for LPS ubiquitination (Otten et al, 2021;Ahel et al, 2020;Ahel et al, 2021). Instead, an adjacent zinc-binding ''RZ'' domain uses a cysteine-dependent mechanism to conjugate ubiquitin, akin to the HECT, RBR, and RCR families of E3 ligases (Otten et al, 2021;Ahel et al, 2021). Recent structural work by the Clausen and Virdee laboratories has described the mechanism of ubiquitin conjugation employed by RNF213 and identified a role for the ATPase core in regulating ligase activity through sensing ATP levels (Ahel et al, 2021).…”

“…Instead, an adjacent zinc-binding ''RZ'' domain uses a cysteine-dependent mechanism to conjugate ubiquitin, akin to the HECT, RBR, and RCR families of E3 ligases (Otten et al, 2021;Ahel et al, 2021). Recent structural work by the Clausen and Virdee laboratories has described the mechanism of ubiquitin conjugation employed by RNF213 and identified a role for the ATPase core in regulating ligase activity through sensing ATP levels (Ahel et al, 2021). Whether this regulatory mechanism plays a role in Salmonella ubiquitination is unclear, considering that Salmonella infection has been shown to induce a Warburg effect on host metabolism, which might decrease cellular ATP levels.…”