System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins. [Display omitted] •High-affinity and selective UbV modulators for 20 HECT E3 ligases•UbV inhibitors hijack the E2 binding site•N-lobe exosite bound UbVs activate HECT E3 ligases•UbVs function in cells and intestinal organoids to reveal new roles of HECT E3 ligases Zhang, Wu et al. generated ubiquitin variants (UbVs) that inhibit or activate the catalytic activities of HECT E3 ligases. These variants can be used to delve into E3 mechanisms and to probe new biological functions of HECT E3s.