Abstract
ADP-ribosyltransferases transfer the ADP-ribose moiety of βNAD+ to an acceptor molecule, usually a protein that modulates the function of the acceptor. Pierisin-1 is an ADP-ribosyltransferase from the cabbage butterfly Pieris rapae and is composed of N-terminal catalytic and C-terminal ricin B-like domains. Curiously, it ADP-ribosylates the DNA duplex, resulting in apoptosis of various cancer cells, which has raised interest in pierisin-1 as an anti-cancer agent. However, both the structure and the mechanism of DNA ADP-ribosylation are unclear. Here, we report the crystal structures of the N-terminal catalytic domain of pierisin-1, its complex with βNAD+, and the catalytic domain with the linker connecting it to the ricin B-like domains. We found that the catalytic domain possesses a defined, positively charged region on the molecular surface but that its overall structure is otherwise similar to those of protein-targeting ADP-ribosyltransferases. Electrophoretic mobility shift assays and site-directed mutagenesis indicated that pierisin-1 binds double-stranded but not single-stranded DNA and that Lys122, Lys123, and Lys124, which are found in a loop, and Arg181 and Arg187, located in a basic cleft near the loop, are required for DNA binding. Furthermore, the structure of the catalytic domain with the linker revealed an autoinhibitory mechanism in which the linker occupies and blocks both the βNAD+- and DNA-binding sites, suggesting that proteolytic cleavage to remove the linker is necessary for enzyme catalysis. Our study provides a structural basis for the DNA-acceptor specificity of pierisin-1 and reveals that a self-regulatory mechanism is required for its activity.
Highlights
ADP-ribosyltransferases transfer the ADP-ribose moiety of NAD؉ to an acceptor molecule, usually a protein that modulates the function of the acceptor
The bacterial mono-ADP-ribosyltransferases form a large family of mono-ADP-ribosylating toxins, including cholera toxin, diphtheria toxin, and pertussis toxin, which target specific cellular proteins to impair the function of the protein (1, 2). 3D structures of various mono ADPribosyltransferases and their complexes with NADϩ have been determined by X-ray crystallography and reveal that the core structures, key residues for catalysis, and NADϩ binding motifs are conserved
Pierisin(1–233)E165Q possesses two -sheets surrounded by six ␣-helices (Fig. 1B). 3 and 4 are twisted relative to each other by 90°
Summary
ADP-ribosyltransferases transfer the ADP-ribose moiety of NAD؉ to an acceptor molecule, usually a protein that modulates the function of the acceptor. Pierisin-1 is an ADP-ribosyltransferase from the cabbage butterfly Pieris rapae and is composed of N-terminal catalytic and C-terminal ricin B–like domains. It ADP-ribosylates the DNA duplex, resulting in apoptosis of various cancer cells, which has raised interest in pierisin-1 as an anti-cancer agent. Unlike the protein ADP-ribosyltransferases mentioned above, pierisin-1 is a unique mono-ADP-ribosyltransferase that targets DNA as the acceptor molecule, with the N-terminal catalytic domain transferring the ADP-ribose moiety to the N2 amino group of guanine (14). To understand the acceptor recognition and catalytic mechanisms of pierisin-1, we determined the crystal structures of the N-terminal catalytic domain of the enzyme with and without a linker (residues 234 –267) (pierisin(1–267) and pierisin(1– 233)) and NADϩ-bound pierisin(1–233) and demonstrate that pierisin-1 has a binding activity toward dsDNA, not ssDNA. We demonstrate that the linker between the N- and C-terminal domains controls catalysis by regulating how NADϩ and DNA access their binding sites
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