Mechanism of E1-E2 interaction for the inhibition of Ubl adenylation.
Conjugates of ubiquitin or its homologues to other proteins occur by strictly ordered steps with ordered addition of substrates for each step. High concentrations of E2 were shown to inhibit the formation of E2∼Ubl thioester and Ubl∼target conjugates. We investigated the mechanism of such inhibitory effect of the SUMO E2 and whether the E2 has two binding sites on its E1, one for the inhibitory effect and one for productive SUMOylation. NMR methods in combination with mutagenesis and biochemical assays revealed that Ubc9 binds to two flexible domains of its free E1 simultaneously, suggesting extensive domain movements in the free E1. Further, interaction of free E1 and E2 inhibits SUMO adenylation, and the interfaces responsible for the inhibition were the same as those required for productive transfer of SUMO from E1 to E2. This study indicates a conformational flexibility-dependent mechanism to control the strictly ordered steps in Ubl modifications.
Wang
Jianghai
J
From the Department of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, California 91010.
Cai
Sheng
S
From the Department of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, California 91010.
Chen
Yuan
Y
From the Department of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, California 91010. Electronic address: ychen@coh.org.
D000249:Adenosine Monophosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D025842:SUMO-1 Protein; C470301:UBA2 protein, human; D000249:Adenosine Monophosphate; D044763:Ubiquitin-Conjugating Enzymes; C470299:SAE1 protein, human; D044764:Ubiquitin-Activating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9