Structural basis of autoinhibition and activation of the DNA-targeting ADP-ribosyltransferase pierisin-1

Abstract ADP-ribosyltransferasestransfer 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-ribosyltransferasefrom the cabbage butterfly Pieris rapaeand is composed of N-terminal catalytic and C-terminal RicinB-like domains. Curiously, it ADP-ribosylatesthe 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-ribosylationare 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 linkerconnecting it to the ricinB-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 assaysand 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 linkerrevealed an autoinhibitory mechanism in which the linkeroccupies and blocks both the βNAD+- and DNA-binding sites, suggesting that proteolytic cleavage to remove the linkeris 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.