Anatomy of the E2 ligase fold: Implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation

Abstract

The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a largescale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis.

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

“…1 B and C) (22)(23)(24). This fold was also related to those of the RWD (RING finger-, WD-repeat-, and DEAD-like proteins) domain and ubiquitin-conjugating enzymes (25,26). The fold similarity between Mad1 CTD and the CTDs of Spc25 and Csm1 was unexpected, as they share no obvious sequence similarity.…”

Enzyme E2 from Chinese White Shrimp Inhibits Replication of White Spot Syndrome Virus and Ubiquitinates Its RING Domain Proteins

“…rFcUbc may enter hemocytes via endocytosis, since it has been reported that recombinant immulectin-3 (IML-3), a C-type lectin from an insect, Manduca sexta, can be translocated from hemolymph into hemocytes (35), a result consistent with ours. It has been reported that the conserved cysteine residue in E2 is involved in the formation of a covalent bond with ubiquitin (1,18,21,32,34). In FcUbc, this conserved Cys is Cys-86, and a mutant FcUbc protein (mFcUbc) in which Cys-86 was mutated to Ser was also expressed and purified.…”

How ubiquitination regulates the TGF‐β signalling pathway: New insights and new players

“…In a recent study addressing the activation of ubiquitin by specific E1-and E2-enzymes, 29 E2s accepted ubiquitin from UBE1 and UBA6 of which 14 were charged by UBE1 (but not by UBA6), 9 were charged by both UBE1 and UBA6 and 1 by UBA6 (but not UBE1), thus highlighting that each E1 has a distinct preference for a specific E2 49 . Within the E2, it is the UBC domain that is of particular importance as it is responsible for specific binding to the relevant E3-ligase 55 Structurally, UBC domains consists of α-helices, β-sheets and variable loop regions that surround an active-site catalytic domain 55 . Amino (N-Ex, N-) and Carboxyl (C-Ex, C-) terminal extensions have also been discovered to flank the catalytic domain ( Figure 2) and shown to be important in steps such as substrate selection, subcellular localisation and dimerisation.…”