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

Abstract

SUMMARY 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.

Structural mechanisms of HECT-type ubiquitin ligases

“…The activities of NEDD4-type enzymes can be modulated by the binding of ubiquitin to an area on the N-lobe, known as the 'exosite' (French et al, 2009;Ogunjimi et al, 2010;Kim et al, 2011;Maspero et al, 2011Maspero et al, , 2013Zhang et al, 2016;French et al, 2017) (Figure 4B). This interaction is mediated by of series of conserved residues on the E3 that contact a region of ubiquitin that includes the canonical 'hydrophobic patch', centered on Ile 44, and extends towards the C-terminus by including Ile 36, Leu 71, and Leu 73 ( Figure 4B, insert).…”

“…This interaction is mediated by of series of conserved residues on the E3 that contact a region of ubiquitin that includes the canonical 'hydrophobic patch', centered on Ile 44, and extends towards the C-terminus by including Ile 36, Leu 71, and Leu 73 ( Figure 4B, insert). The affinity of ubiquitin for the exosite falls into the micromolar K D -range in vitro (Kim et al, 2011;Maspero et al, 2011;Zhang et al, 2016;French et al, 2017).…”

“…Recently, ubiquitin variants with enhanced affinities for the exosite were employed to dissect the role of the exosite in several HECT E3 enzymes (Zhang et al, 2016). These comparative studies demonstrated that the occupation of the exosite with ubiquitin variants has rather complex effects with regard to processivity, chain elongation, target lysine choice, and domain-domain interactions and can impact individual parts of the reaction in different ways, depending on the identities of the E3 and target proteins and precise constructs used (Zhang et al, 2016).…”

Proteostasis in Huntington’s Disease: Disease Mechanisms and Therapeutic Opportunities

“…In the case that high quality chemical tools are not available, we and our collaborative labs have developed highly specific and potent proteinaceous biological binder, ubiquitin variants, to interfere with the ubiquitin regulatory enzymes 149,150 for target validation purpose. Use of such tool molecules will help identify and validate therapeutic targets in the HTT proteostatis pathway.…”

Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21

“…Moreover, interpretation of the results in the context of the human USP family shows that many conserved functional interactions likely are generalizable to most family members. We have recently reported structures of additional Ubvs in complex with E3 ligases of the Homologous to E6AP C terminus (HECT), Skp1 Cul1 F-box (SCF), and Really Interesting New Gene (RING) families (24,25), and the extension of the methods described here to these and other types of Ub-protein interactions may identify commonalities and differences in how Ub recognizes diverse structural folds to mediate biological effects.…”