Role of the Zn(2+) motif of E1 in SUMO adenylation.
Post-translational modifications by ubiquitin-like proteins are among the most important mechanisms for regulating a wide variety of cellular functions. In these modifications an E1 enzyme activates each ubiquitin-like protein (Ubl) by adenylation of the Ubl C-terminal COOH group and then forms a thioester bond with the adenylated C-terminal COOH group of the Ubl. Previous x-ray crystallography studies revealed a conserved zinc motif in the SUMO and NEDD8 E1; however, the function of this Zn(2+) motif is unclear. In this study, using quantitative ATP:PPi isotope exchange assays in combination with site-directed mutagenesis, we show that the conserved Zn(2+) motif in the SUMO E1 is important for SUMO adenylation and is critical for the E1 pseudo-ordered substrate binding mechanism. Furthermore, Zn(2+) motif mutants showed significantly reduced k(cat) values for ATP:PPi isotope exchange assays, suggesting that the Zn(2+) motif is important in binding and preventing SUMO adenylate from dissociating from E1 before formation of the thioester conjugate. Because the Zn(2+) motif is located in a cross-over loop that is known to have conformational flexibility, the results described here suggest that this cross-over loop interacts with Ubl in the multistep, dynamic process of Ubl activation by E1s.
Wang
Jianghai
J
Division of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Chen
Yuan
Y
D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D007554:Isotopes; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D025801:Ubiquitin / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
D007554:Isotopes; D025842:SUMO-1 Protein; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D015032:Zinc