# E2 variants for probing E3 ubiquitin ligase activities

## TL;DR

Researchers engineered E2 enzyme variants to better study E3 ubiquitin ligase functions and interactions in cells.

## Contribution

A generalizable method to engineer E2 variants with enhanced selectivity and affinity for specific E3 ligases is introduced.

## Key findings

- E2 variants (E2Vs) were developed to distinguish E3 ligase functions in multi-E3 complexes.
- Cryo-EM structures of RBR E3 ligases in substrate-bound complexes were obtained using E2Vs.
- E2Vs enabled profiling of endogenous RBR E3 responses to extracellular signals.

## Abstract

E2 ubiquitin conjugating enzymes and E3 ligases regulate virtually all aspects of eukaryotic cell biology. Despite progress in interrogating functions of these enzymes, understanding their regulation remains challenging due to a paucity of probes for E3 enzymes, and because of E2 promiscuity, since each E2 functions with many E3s. Also, E2–E3 interactions are typically of moderate affinity. Here, focusing on UBE2L3 and UBE2D3 E2s and RING-between-RING (RBR)-type E3s, we present a generalizable approach for engineering E2 variants (E2Vs) with enhanced selectivity and affinity toward particular E3 ligases. Engineered E2Vs allow dissecting E3 functions in a multi-E3 complex, determining E2–E3 complex structures, and probing E3s responding to cellular signals.

E3 ligases partner with E2 enzymes to regulate vast eukaryotic biology. The hierarchical nature of these pairings, with >600 E3s and ~40 E2s in humans, necessitates that E2s cofunction with numerous different E3s. Here, focusing on E3s in the RING-between-RING (RBR) family and their partner UBE2L3 and UBE2D-family E2s, we report an approach to interrogate selected pathways. We screened phage-displayed libraries of structure-based E2 variants (E2Vs) to discover enzymes with enhanced affinity and specificity toward half of all RBR E3 ligases (ARIH1, ARIH2, ANKIB1, CUL9, HOIL1, HOIP, and RNF14). Collectively, these E2Vs allowed distinguishing actions of different cofunctioning E3s, obtaining high-resolution cryogenic Electron Microscopy (cryo-EM) structures of an RBR E3 in the context of a substrate-bound multiprotein complex, and profiling an endogenous RBR E3 response to an extracellular stimulus. Overall, we anticipate that E2V technology will be a generalizable tool to enable in-depth mechanistic and structural analysis of E3 ligase functions, and mapping their activity states and protein partners in cellular signaling cascades.