title: The N-terminal extension of UBE2E ubiquitin-conjugating enzymes limits chain assembly.
abstract: Protein ubiquitylation depends upon the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). All E2s have a conserved ubiquitin-conjugating (UBC) domain but many have variable extensions N- and C-terminal to the UBC domain. For many E2s, the function of the extension is not well understood. Here, we show that the N-terminal extension of the UBE2E proteins regulates formation of polyubiquitin chains by the processive UBC domain. Target proteins are therefore monoubiquitylated by full-length UBE2E, whereas the UBC domain alone polyubiquitylates proteins. Although the N-terminal extension of UBE2E1 is largely disordered in solution, these residues have a critical role in limiting chain building, and when fused to the highly processive E2, UBE2D2, ubiquitylation is limited. For some E2s, interaction of ubiquitin with the 'backside' of the UBC domain promotes polyubiquitylation. However, interaction of ubiquitin with the backside of the UBC domain of UBE2E1 does not appear to be important for processivity. This study underscores the importance of studying full-length E2 proteins and not just the highly conserved core domain.
authors.affiliation: Biochemistry Department, University of Otago, Dunedin 9054, New Zealand.
authors.forename: Frances-Rose
authors.initials: FR
authors.lastname: Schumacher
authors.forename: Georgina
authors.initials: G
authors.lastname: Wilson
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Day
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D016415:Sequence Alignment; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes
keywords: CARP2; E1; E2; E3; GST; K0; MALS; MDM2; PBS; RING; SEC; UBA domain; UBC domain; WT; cIAP2; caspase-8 and -10 associated RING protein 2; cellular inhibitor of apoptosis protein 2; chain formation; glutathione S-transferase; lysine-zero; mouse double minute 2; multi-angle light scattering; phosphate-buffered saline; size-exclusion chromatography; ubiquitin; ubiquitin ligase; ubiquitin-activating enzyme; ubiquitin-associated domain; ubiquitin-conjugating domain; ubiquitin-conjugating enzyme; wild type