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Article Dans Une Revue Journal of Biological Chemistry Année : 2023

Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic

Résumé

Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesised through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9∼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9∼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9∼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2∼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterise the resulting mimetic of human UBC9∼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9–SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerisation via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9∼SUMO in some cellular contexts.
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Dates et versions

hal-04134078 , version 1 (20-06-2023)

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Stéphane Goffinont, Franck Coste, Pierre Prieu-Serandon, Lucija Mance, Virginie Gaudon, et al.. Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic. Journal of Biological Chemistry, In press, ⟨10.1016/j.jbc.2023.104870⟩. ⟨hal-04134078⟩
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