Common Mechanism for Target Specificity of Protein- and DNA-Targeting ADP-Ribosyltransferases

Abstract

Many bacterial pathogens utilize ADP-ribosyltransferases (ARTs) as virulence factors. The critical aspect of ARTs is their target specificity. Each individual ART modifies a specific residue of its substrates, which could be proteins, DNA, or antibiotics. However, the mechanism underlying this specificity is poorly understood. Here, we review the substrate recognition mechanism and target residue specificity based on the available complex structures of ARTs and their substrates. We show that there are common mechanisms of target residue specificity among protein- and DNA-targeting ARTs.

Keywords: ADP-ribosyltransferase; complex structure of enzyme and substrate; substrate recognition; target residue specificity.

Figure 1

Comparison of R-S-E class, H-Y-E class, and other ADP-ribosyltransferases. (a) Substrate, target residue, and conserved motifs. Conserved R-S-E and H-Y-E residues are shown in red. In the R-S-E motif, the third aromatic residue and the sixth glutamate or glutamine residue in the ADP-ribosylating toxin turn-turn (ARTT) loop (X-X-ϕ-X-X-E/Q-X-E) are shown in orange and blue, respectively. ADP-ribosyltransferases (ARTs) with available substrate-complexed structures are indicated with asterisks. (b) Superimposed structures of the ART domains of the 11 ARTs are shown in (d). The structure of the ART core, including six strands and the helix, are shown in violet. (c) Close-up views of ART core structures. Individual strands are shown in different colors and are numbered from the N-terminus to the C-terminus. NAD bound to Ia is shown as a ball-and-stick model. (d) Structures of the ART domain. ART core structures are shown in violet. R-S-E and H-Y-E residues are shown as sphere models. NAD or NAD analogs are shown as stick models. Protein Data Bank (PDB) IDs: Ia, 4h03; C3, 4xsh; PT, 1bcp; SdeA, 5yij; Pierisin, 5h6j; ScARP, 5zj5; ExoA, 2zit; Arr, 2hw2; PARP2, 6tx3; ParT, 6d0h; Tpt1, 6e3a.

Figure 2

Comparison of complex structures of ARTs and their substrates. (a) R-S-E class ARTs. Top, overall structures; middle, close-up views of active sites; bottom, interactions between the target and the sixth amino acid of the ARTT loop. (b) H-Y-E class ARTs. Top, overall structures; bottom, close-up views of active sites. The N-terminal domain of Ia and the α-helical domain of SdeA are shown in light green color. The ARTT loops of R-S-E class ARTs are shown in orange. The NC1 of NAD (electrophile) and the modified atom (nucleophile) are shown as sphere models. Distances between two atoms involved in forming the new bond, namely the NC1 of NAD (electrophile) and the modified atom (nucleophile), are shown in red. Black circles indicate the third catalytic glutamate residues of the R-S-E and H-Y-E motifs. (c) Positional relationships between nucleophiles and NADs. Superimposition of six complex structures are shown in (a,b). R-S-E and H-Y-E residues are shown as stick models. NADs and targets are shown as lines. NC1 atoms of NADs (electrophiles) and modified atoms (nucleophile) are shown as sphere models. PDB IDs: Ia–actin, 4h03; C3–RhoA, 4xsh; SdeA–Ub, 5yij; ScARP–GDP, 5zj5; ExoA–eEF2, 2zit; Arr–rifampin, 2hw2.