A Stable Chemical SUMO1-Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2-E3 Ligase Complex

Ubiquitin and ubiquitin‐like (Ubl) modifiers such as SUMO are conjugated to substrate proteins by E1, E2, and E3 enzymes. In the presence of an E3 ligase, the E2∼Ubl thioester intermediate becomes highly activated and is prone to chemical decomposition, thus making biochemical and structural studies difficult. Here we explored a stable chemical conjugate of the E2 enzyme from the SUMO pathway, Ubc9, with its modifier SUMO1 as a structural analogue of the Ubc9∼SUMO1 thioester intermediate, by introducing a triazole linkage by biorthogonal click chemistry. The chemical conjugate proved stable against proteolytic cleavage, in contrast to a Ubc9–SUMO1 isopeptide analogue obtained by auto‐SUMOylation. Triazole‐linked Ubc9–SUMO1 bound specifically to the preassembled E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9, thus suggesting that it is a suitable thioester mimic. We anticipate interesting prospects for its use as a research tool to study protein complexes involving E2 and E3 enzymes. Chemical camouflage: Ubiquitin and SUMO bound as thioesters to their E2 conjugating enzymes are labile intermediates in the presence of an E3 ligase, a fact that hampers biochemical characterization of the charged E2/E3 complexes. A stable chemical conjugate of SUMO1 with the SUMO E2 enzyme is now reported as a thioester mimic to address these limitations.