Crystal structure of a fragment of mouse ubiquitin-activating enzyme.
Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a four-helix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitin-like modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine half-domains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8- and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.
Szczepanowski
Roman H
RH
International Institute of Molecular and Cell Biology, ul. Trojdena 4, 02-109 Warsaw, Poland.
Filipek
Renata
R
Bochtler
Matthias
M
D020816:Amino Acid Motifs; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D002845:Chromatography; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D000075687:NEDD8 Protein; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry*; D014452:Ubiquitins / Q000737:chemistry
D046912:Multiprotein Complexes; D000075687:NEDD8 Protein; C513172:Nedd8 protein, mouse; D025842:SUMO-1 Protein; D025801:Ubiquitin; D014452:Ubiquitins; D044764:Ubiquitin-Activating Enzymes; D003545:Cysteine