Multi-tiered pairing selectivity between E2 ubiquitin-conjugating enzymes and E3 ligases.

Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin-conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyze the attachment of ubiquitin and have emerged as key mediators of chain assembly. They are largely responsible for the type of linkage between ubiquitin moieties and thus, the fate endowed onto the modified substrate. However, E2-E3 pairing remains largely unexplored. We therefore interrogated the interaction selectivity between 37 E2s and PUB22, a U-box type E3 ubiquitin ligase that is involved in the dampening of immune signaling. We show that whereas the U-box domain, which mediates E2 docking, is able to interact with 18 of 37 tested E2s, the substrate interacting armadillo (ARM) repeats impose a second layer of specificity, allowing the interaction with 11 E2s. activity assayed by autoubiquitination only partially recapitulated the selectivity. Moreover, pairing was modulated during the immune response; pairing with group VI UBC30 was inhibited, whereas interaction with the K63 chain-building UBC35 was increased. Functional analysis of mutants shows that they partially mimic enhanced activation of immune responses. Together, our work provides a framework to interrogate E2-E3 pairing and reveals a multi-tiered and dynamic E2-E3 network.