Alternative allosteric mechanisms can regulate the substrate and E2 in SUMO conjugation

Sumoylation is the covalent attachment of small ubiquitin-like modifier (SUMO) to a target protein. Similar to other ubiquitin-like pathways, three enzyme types are involved that act in succession: an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). To date, unlike other ubiquitin-like mechanisms, sumoylation of the target RanGAP1 (Target^RanGAP1) does not absolutely require the E3 of the system, RanBP2 (E3^RanBP2), since the presence of E2 (E2^Ubc9) is enough to sumoylate Target ^RanGAP1. However, in the presence of E3, sumoylation is more efficient. To understand the role of the target specificity of E3 ^RanBP2 and E2^Ubc9, we carried out molecular dynamics simulations for the structure of E2^Ubc9-SUMO-Target^RanGAP1 with and without the E3^RanBP2 ligase. Analysis of the dynamics of E2^Ubc9-SUMO-Target^RanGAP1 in the absence and presence of E3^RanBP2 revealed that two different allosteric sites regulate the ligase activity: (i) in the presence of E3^RanBP2, the E2 ^Ubc9's loop 2; (ii) in the absence of E3^RanBP2, the Leu65-Arg70 region of SUMO. These results provide a first insight into the question of how E3^RanBP2 can act as an intrinsic E3 for E2 ^Ubc9 and why, in its absence, the activity of E2^Ubc9-SUMO- Target^RanGAP1 could still be maintained, albeit at lower efficiency.