A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes. [Display omitted] •Identification of selective UbVs that inhibit or activate RING/U-box E3 ligases•Structural and biochemical analyses reveal mechanisms of modulation by UbVs•UbVs inhibit CBL and UBE4B by blocking the E2∼Ub binding site•A UbV dimer stimulates XIAP activity by stabilizing E2∼Ub Gabrielsen et al. report the identification of selective ubiquitin variants that inhibit or activate the catalytic domains of RING/U-box E3 ligases. Inhibition is achieved by blocking the E2∼ubiquitin binding surface of RING/U-box domains, whereas activation is accomplished by stabilizing the primed E2∼ubiquitin conformation.