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.
Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Strasse 2, 48149 Münster (Germany).
Stefanie
S
Sommer
Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg (Germany).
Tobias
T
Ritterhoff
Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg (Germany). f.melchior@zmbh.uni-heidelberg.de.
Frauke
F
Melchior
Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Strasse 2, 48149 Münster (Germany). henning.mootz@uni-muenster.de.
Henning D
HD
Mootz
D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D006868:Hydrolysis; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D059748:Proteolysis; D025842:SUMO-1 Protein / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014230:Triazoles / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D000480:Alkynes; D001386:Azides; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D025842:SUMO-1 Protein; D013438:Sulfhydryl Compounds; D014230:Triazoles; C094493:ran-binding protein 2; D003300:Copper; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9
SUMO; biological activity; click chemistry; enzyme catalysis; post-translational modifications; ubiquitin