Certain RING ubiquitin ligases (E3s) dimerize to facilitate ubiquitin (Ub) transfer from ubiquitin-conjugating enzyme (E2) to substrate, but structural evidence on how this process promotes Ub transfer is lacking. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub (UbcH5B∼Ub). The structure reveals extensive noncovalent donor Ub interactions with UbcH5B and both subunits of the RING domain dimer that stabilize the globular body and C-terminal tail of Ub. Mutations that disrupt these noncovalent interactions or RING dimerization reduce UbcH5B∼Ub binding affinity and ubiquitination activity. Moreover, NMR analyses demonstrate that BIRC7 binding to UbcH5B∼Ub induces peak-shift perturbations in the donor Ub consistent with the crystallographically-observed Ub interactions. Our results provide structural insights into how dimeric RING E3s recruit E2∼Ub and optimize the donor Ub configuration for transfer.

RING E3 ligases catalyze the transfer of ubiquitin (Ub) from E2 ubiquitin-conjugating enzyme thioesterified with Ub (E2~Ub) to substrate. For RING E3 dimers, the RING domain of one subunit and tail of the second cooperate to prime Ub, but how this is accomplished by monomeric RING E3s in the absence of a tail-like component is unknown. Here, we present a crystal structure of a monomeric RING E3, Tyr363-phosphorylated human CBL-B, bound to a stabilized Ub-linked E2, revealing a similar mechanism in activating E2~Ub. Both pTyr363 and the pTyr363-induced element interact directly with Ub’s Ile36 surface, improving the catalytic efficiency of Ub transfer by ~200-fold. Hence, interactions outside the canonical RING domain are crucial for optimizing Ub transfer in both monomeric and dimeric RING E3s. We propose that an additional non-RING Ub-priming element may be a common RING E3 feature.

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RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.