A Mechanistic View of the Role of E3 in Sumoylation

Sumoylation, the covalent attachment of SUMO (Small Ubiquitin-Like Modifier) to proteins, differs from other Ubl (Ubiquitin-like) pathways. In sumoylation, E2 ligase Ubc9 can function without E3 enzymes, albeit with lower reaction efficiency. Here, we study the mechanism through which E3 ligase RanBP2 triggers target recognition and catalysis by E2 Ubc9. Two mechanisms were proposed for sumoylation. While in both the first step involves Ubc9 conjugation to SUMO, the subsequent sequence of events differs: in the first E2-SUMO forms a complex with the target and E3, followed by SUMO transfer to the target. In the second, Ubc9-SUMO binds to the target and facilitates SUMO transfer without E3. Using dynamic correlations obtained from explicit solvent molecular dynamic simulations we illustrate the key roles played by allostery in both mechanisms. Pre-existence of conformational states explains the experimental observations that sumoylation can occur without E3, even though at a reduced rate. Furthermore, we propose a mechanism for enhancement of sumoylation by E3. Analysis of the conformational ensembles of the complex of E2 conjugated to SUMO illustrates that the E2 enzyme is already largely pre-organized for target binding and catalysis; E3 binding shifts the equilibrium and enhances these pre-existing populations. We further observe that E3 binding regulates allosterically the key residues in E2, Ubc9 Asp100/Lys101 E2, for the target recognition.Author Summary: Post-translational modifications constitute key regulatory mechanisms in the cell. One of these modifications is the tagging of the target protein with a smaller molecule. SUMO is such a ubiquitin-like tag protein, and sumoylation is the process of tagging proteins with SUMO. The malfunctioning of sumoylation is linked with diseases such as Alzheimer's, Parkinson's, and cancer. Based on experimental observations, two paths were suggested for sumoylation, the first and more efficient involves the E1, E2 and E3 enzymes; the second only the E1 and E2. Here we investigate these alternative paths of sumoylation. Our results offer an explanation for how sumoylation can take place with only the E1 and E2 enzymes, and for the mechanistic role of E3. They emphasize that E2 bound to SUMO is already pre-organized for the transfer of SUMO to a target protein and E3 binding further stabilizes the conformations, shifting the ensemble and thus increasing the efficiency of the sumoylation.