title: Systematic determinations of SUMOylation activation intermediates and dynamics by a sensitive and quantitative FRET assay.
abstract: Ubiquitination and SUMOylation are multi-step cascade reactions, in which small protein modifiers are activated by E1 activating enzyme, transferred to E2 conjugating enzyme, and conjugated to substrates mediated by the E3 ligase in vivo. The structural and biochemical bases for the cascade reactions have been elucidated by several studies. However, the reaction intermediates and dynamics of these peptide modifiers among the enzymes have not been completely elucidated. Here we report detailed investigations of SUMOylation dynamics and interaction switches of SUMO1 among its ligases using FRET technology. These studies show that, while SUMO1 and the E1 subunit Aos1 or Uba2 have no intrinsic affinity for each other, the adenylation of SUMO1 carried out by Aos1 requires the presence of Uba2, and subsequently conformational changes trigger the interaction of SUMO1 and Uba2 for a thioester bond formation. The reaction intermediates among SUMO1 and its ligases are indirectly revealed by FRET signals generated by each pair. Furthermore, the transfer of SUMO1 from Uba2 to E2 enzyme, Ubc9, depends on the formation of a thioester bond between SUMO1 and Ubc9, and requires non-covalent interaction between Ubc9 and Uba2, but not between Ubc9 and SUMO1. These interaction switches provide the physical and biochemical bases for the SUMO activation and a transfer cascade required for SUMO activation.
authors.affiliation: Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, Riverside, CA 92521, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.forename: Jiayu
authors.initials: J
authors.lastname: Liao
mesh_terms: D000465:Algorithms; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D025842:SUMO-1 Protein / Q000737:chemistry* / Q000378:metabolism; D058207:Sumoylation / Q000502:physiology*; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D044764:Ubiquitin-Activating Enzymes