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 large-scale 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.

Fig. 1. Multiple alignment of selected E2 domains, representing active site variants

Sequences are shown with their gene name, species abbreviations, and genbank ID (gi) numbers separated by underscores given to the left of the alignment. PDB ids of sequences with solved X-ray crystal structures are highlighted in orange. The top line of the alignment labels the major conserved features of the fold and the line below the alignment shows the consensus secondary structure; E and H denote β-strand and α-helix, respectively. Family names are listed to the right of the alignment. The bacterial families A–E correspond to representatives of rows 6A–6E in Table 2 of (Iyer et al., 2006). Columns in the alignment are colored based on the functional role of the residue: the catalytic cysteine is colored in yellow and shaded in red, flap histidine and asparagine residues and other residues predicted to perform analogous roles are colored in red and shaded in yellow, the proline and hydrophobic residue forming a conserved stabilizing contact are shaded in green, and the conserved aromatic residue and other residues predicted to form a chain of interacting residues are colored in gray. Species abbreviations are as follows: Afum, Aspergillus fumigatus; Amel, Apis mellifera; Asp., Azoarcus sp.; Atha, Arabidopsis thaliana; Bcer, Bacillus cereus; Bvie, Burkholderia vietnamiensis; Cele, Caenorhabditis elegans; Cneo, Cryptococcus neoformans; Cper, Clostridium perfringens; Ddis, Dictyostelium discoideum; Dmel, Drosophila melanogaster; Ecol, Escherichia coli; Ehis, Entamoeba histolytica; Glam, Giardia lamblia; Goxy, Gluconobacter oxydans; Gura, Geobacter uraniumreducens; Hsap, Homo sapiens; Lmaj, Leishmania major; Maqu, Marinobacter aquaeolei; Mdeg, Microbulbifer degradans; Mmus, Mus musculus; Otau, Ostreococcus tauri; Parc, Psychrobacter arcticus; Pber, Parvularcula bermudensis; Pfal, Plasmodium falciparum; Pnap, Polaromonas naphthalenivorans; Ppro, Pelobacter propionicus; Psp., Polaromonas sp.; Ptet, Paramecium tetraurelia; Reut, Ralstonia eutropha; Rmet, Ralstonia metallidurans; Rsp., Rhizobium sp.; Scer, Saccharomyces cerevisiae; Spom, Schizosaccharomyces pombe; Tann, Theileria annulata; Tbru, Trypanosoma brucei; Tcru, Trypanosoma cruzi; Tvag, Trichomonas vaginalis; Vcho, Vibrio cholerae; Xaxo, Xanthomonas axonopodis.

Fig. 2. Topology diagram of generalized E2 domain and tree depicting general relationships between E2 domain families

(A) Cartoon representation showing conserved elements of the E2 fold. The extended elements of the flap are colored in gray. Labels for all elements in the figure correspond to those in the text. Locations of the flap histidine and flap asparagine in the classic E2 active site are shown as circles colored in yellow, while other conserved residues mentioned in the text are depicted as line drawings. The C-terminal Ub/Ubl tail that forms hydrogen-bonding interactions with the C-terminal extended element is depicted as a brown colored arrow. The red lines represent hydrogen bonding occurring before attack by the target lysine. (B) Reconstructed evolutionary history of the E2 protein fold. Lineages in the fold are listed to the right, with inferred evolutionary depth traced by solid horizontal lines across the relative temporal epochs representing key evolutionary transition periods shown as vertical lines. Horizontal lines are color-coded according to observed phyletic distribution in a given lineage, red applied to bacteria-specific lineages and green applied to eukaryote-specific lineages. Dashed lines indicate uncertainty regarding the origins of a lineage. Yellow circles placed on a horizontal line indicate a lineage has lost catalytic activity.

Fig. 3. Surface diagrams displaying conserved binding grooves

Molecular surfaces are shown for the two conserved binding grooves in selected members of the E2 fold. The different grooves are labeled at the top of the figure, the ubiquitin-binding groove is on the left and the putative E1-binding groove is on the right. Two surface depictions in the same orientation are provided for each groove, the left surface provides an opaque view of the surface with the catalytic cysteine and flap asparagine/histidine residues colored in yellow, with family-specific conserved residues associated with the groove colored blue. The right surface provides cartoon depictions of the secondary structure of the domain colored in yellow framed by the transparent outline of the molecular surface. The catalytic cysteine and flap asparagine/histidine (or equivalent residues) are rendered as ball-and-sticks and colored in red. The surfaces of other conserved residues in a family are colored blue. Family names and PDB codes are given at the left.

Fig. 4. Cartoon structural diagrams showing E2 domain active site variants

Cartoon representations of X-ray structures are shown with PDB codes and family names provided at the left. Strands are colored in blue and helices in red. The catalytic cysteine and the flap asparagine/histidine or predicted equivalent residues are labeled and rendered as ball-and-sticks colored in yellow. The molecular surfaces of these residues, as well as residues predicted to form the conserved interacting chain are rendered using their van der Waal radii. The histidine residue predicted to be important in the Ubc6 family, but not observed in the crystal structure due to structure disorder is placed in its approximate predicted spatial location and colored pink. The proline and hydrophobic resides forming a conserved hydrophobic contact are rendered as ball-and-sticks and are colored orange. The active versions of the domain are boxed in orange and the inactive UEV1 family boxed in purple.