The N-Terminal Extension of UBE2E Ubiquitin-Conjugating Enzymes Limits Chain Assembly

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. [Display omitted] •Background: E2 conjugating enzymes have a central role in the ubiquitylation cascade and can determine the architecture of ubiquitin modifications. Some E2s comprise just the conserved core domain, whereas others have N- and C-terminal extensions.•Result: Here, we show that the ubiquitin-conjugating domain of UBE2E proteins can build polyubiquitin chains, but the N-terminal extension of this family of E2 proteins limits polyubiquitylation. As a consequence, the unmodified full-length proteins preferentially monoubiquitylate target proteins.•Significance: The UBE2E family of proteins interact with numerous E3 ligases, including many that also interact with the chain building UBE2D family; therefore, the selective recruitment of these two E2s may influence the nature of the modification attached to target proteins.