Role of NEDD8 in CUL2‐dependent ubiquitination of substrates with distinct sizes

As the largest family of ubiquitin E3 ligases in eukaryotes, cullin‐RING ligases (CRLs) play essential roles in a myriad of biological processes by catalyzing the ubiquitination of key regulatory proteins. Neddylation, the conjugation of NEDD8 to cullins, is one of the most important mechanisms regulating the activity of CRLs. Biochemical studies have demonstrated that neddylation promotes the activity of CUL1‐based CRL1s, leading to increased rates of substrate ubiquitination. Structural studies further suggested that neddylation triggered a conformational change that potentially bridged a gap between the donor ubiquitin and the acceptor substrate. These findings led to a hypothesis that the ubiquitination of larger CRL substrates may depend less on neddylation because the substrates could close the gap by themselves. Here, we explored the effect of neddylation on CRL2 VHL ‐dependent ubiquitination of substrates with distinct sizes, through ex vivo degradation and in vitro ubiquitination assays. We first found that treating HeLa or HEK293 cells with pevonedistat, a small molecule drug inhibiting neddylation, reduced the degradation rates of small‐sized p27 or the ODD domain of HIF1α, but not the full‐length HIF1α. Though results from these initial trials support our hypothesis, we further found that inhibiting neddylation did reduce the degradation rate of the full‐length HIF1α in RCC4 cells expressing exogenous VHL, in which CRL2 VHL ‐dependent ubiquitination is the primary mechanism for HIF1α degradation. This result suggests that the degradation of HIF1α we observed in HEK293 cells in the presence of pevonedistat was CRL2 VHL ‐independent. To directly test our hypothesis, we reconstituted CRL2 VHL substrate ubiquitination in vitro and found that neddylation promotes the ubiquitination of the full‐length HIF1α and the degron peptide of HIF1α to similar extents, demonstrating that neddylation activates CRL2 VHL ‐dependent ubiquitination regardless of the substrate sizes. Our findings are consistent with the recently updated structural model of the mechanism for NEDD8‐induced CRL activation, suggesting that neddylation not only brings the acceptor substrate in close proximity to the donor ubiquitin, but also tunes the CRL conformation to ensure efficient ubiquitination of different‐sized substrates.