Immortalized Mouse Bone Marrow Macrophages-SV40

Structural Mechanisms of Calmodulin Activation of Shigella Effector Ospc3 to ADP-Riboxanate Caspase-4/11 and Block Pyroptosis

Pyroptosis is a programmed cell death essential for antibacterial immunity that is triggered by the caspase-4/11-GSDMD axis. OspC3 effector from Shigella flexneri antagonizes pyroptosis through ADP-riboxanation of caspase-4/11. However, the structural basis by which calmodulin activates OspC3 to catalyze this modification and inhibit pyroptosis remains unclear.

Here, Huo et al. identified Ca²⁺-free calmodulin (CaM) that binds and activates the ADP-riboxanase activity of OspC3. The crystal structures of OspC3–CaM and OspC3–caspase-4 complexes, as well as the CaM–OspC3–caspase-4 ternary complex, reveal the unique binding of CaM to the N-terminal domain of OspC3, specific recognition of caspase-4 by the ankyrin repeat domain of OspC3, and NAD⁺ binding-triggered reorganization of the catalytic pocket. Within this pocket, D231 and D177 of OspC3 activate the substrate arginine for ADP-ribosylation and downstream deamination. Shigella infection was used to validate the functional significance of our structural discoveries. In S. flexneri, ΔospC3 triggered GSDMD cleavage and pyroptosis in human A431 cells and mouse iBMMs, which was mitigated by re-expression of WT OspC3 (Fig. 1a). In contrast, mutants defective in caspase-4/11 recognition (L414D/V449D), CaM binding (V59D/F62D/L63D), or NAD⁺ loading and arginine activation (F188A/F211A, E326A, D231A) failed to block the caspase-4/11-GSDMD axis (Fig. 1a). The ΔospC3 strain exhibited a replication defect in mouse livers and spleens, which permitted mouse survival (Fig. 1b and c). These OspC3 mutants, in contrast to WT OspC3, failed to rescue this defect of ΔospC3 in mouse tissues and similarly permitted mouse survival like ΔospC3-infected mice (Fig. 1b and c). These results validate the functional relevance of CaM binding and caspase-4/11 recognition for OspC3 activation.

Fig. 1. Functional analyses of structural mechanisms for CaM activation of OspC3 to target and ADP-riboxanate caspase-4/11 (Huo Y J, Zeng H, et al., 2023).