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

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 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 Lys, Lys, and Lys, which are found in a loop, and Arg and Arg, 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.

Dynamics of Scabin toxin. A proposal for the binding mode of the DNA substrate

“…In the Scabin·NAD + ·DNA ternary complex ( Fig 8a ), the active conformation of Scabin reveals a smaller structural difference between the apo-Scabin (5DAZ) structure and the Scabin·NADH complex (5TLB), with RMSDα values of 0.88 Å and 0.95 Å, respectively. Regarding the active conformation of the NAD + , its binding pose is essentially identical to that of NADH in the Scabin·NADH complex (5TLB) and to that of NAD + in the Pierisin-1·NAD + complex (5H6J), except for the dihedral P N -O–C-C [ 17 ].…”

“…Furthermore, Scabin has low sequence identity with most mART toxins except for the Pierisin subgroup and significantly differs in topology from well characterized mART toxins like iota and C3-group members, which are dominated by high helical content at the N-terminus [ 15 ]. Scabin shares structural homology with Mosquitocidal toxin (MTX) from Bacillus sphaericus [ 16 ] and the apoptosis-inducing Pierisin-1 from the cabbage butterfly Pieris rapae [ 17 ]. The roles of the ARTT and PN loops are well defined in mART toxins [ 18 , 19 ].…”

“…The roles of the ARTT and PN loops are well defined in mART toxins [ 18 , 19 ]. However, the location and topology of the α 1 -α 2 motif is unique for the Pierisin-like subgroup of the CT group of mART toxins with known structures [ 9 , 17 ]. Thus, it is important to consider the role of the α 1 -α 2 motif in the dynamic and catalytic properties of Pierisin-like toxins beyond its obvious structural role.…”

Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP

“…However, the present complex structure provides an important insight into the common guanine specificity in the pierisin family. Recently the structure of pierisin-1 was revealed (31). In pierisin-1, the key residues (Trp160, Gln163, and Glu165 on the ARTT-loop) are also conserved.…”

“…S3), suggesting a difference in substrate preference as described later in the discussion. In pierisin, the long PN-loop and basic cleft were shown to be important for dsDNA by a mutational study (31). Scabin structures were first revealed in apo-and inhibitor-bound forms (32) and very recently in the NADH-bound form (33).…”