A nanobody that binds to the backside of the ubiquitin conjugating enzyme Ube2G2 differentially affects interactions with its partner E3 Ligases

Ubiquitin conjugating E2 enzymes are a set of ~40 proteins that play a central role in the ubiquitination cascade. They transfer ubiquitin from an E1 enzyme to substrates with the help of an E3 enzyme. The members of the E2 family share structural similarity in their conserved UBC fold. This complicates an assessment of the specificity of E2-E3 interactions. We identified a nanobody that binds to the 'backside' region of Ube2G2, an E2 involved in ER protein quality control. This binding does not affect ubiquitin loading but shows varying degrees of inhibition on E3-mediated ubiquitination, in the order HRD1 > CHIP >> TRC8. A naturally occurring segment that binds Ube2G2's backside, referred to as G2BR (Ube2G2 Binding Region), shows a similar inhibitory effect depending on the identity of the interacting E3. The G2BR in the Ube2G2-cognate E3 Gp78 enhances Ube2G2's activity, but its deletion results in a similar inhibition upon addition of the nanobody. Occupation of a single binding site on an E2 can thus affect its interactions with different E3s.