Abstract
The caspase-4/11-GSDMD pyroptosis axis recognizes cytosolic lipopolysaccharide for antibacterial defenses. Shigella flexneri employs an OspC3 effector to block pyroptosis by catalyzing NAD+-dependent arginine ADP-riboxanation of caspase-4/11. Here, we identify Ca2+-free calmodulin (CaM) that binds and stimulates OspC3 ADP-riboxanase activity. Crystal structures of OspC3-CaM and OspC3-caspase-4 binary complexes reveal unique CaM binding to an OspC3 N-terminal domain featuring an ADP-ribosyltransferase-like fold and specific recognition of caspase-4 by an OspC3 ankryin repeat domain, respectively. CaM-OspC3-caspase-4 ternary complex structures show that NAD+ binding reorganizes the catalytic pocket, in which D231 and D177 activate the substrate arginine for initial ADP-ribosylation and ribosyl 2'-OH in the ADP-ribosylated arginine, respectively, for subsequent deamination. We also determine structures of unmodified and OspC3-ADP-riboxanated caspase-4. Mechanisms derived from this series of structures covering the entire process of OspC3 action are supported by biochemical analyses in vitro and functional validation in S. flexneri-infected mice.