Abstract

Caspase-4 drives non-canonical inflammasome signaling by cleaving gasdermin D (GSDMD) to trigger pyroptosis. Cleavage of the interdomain linker (IDL) in caspase-4 yields distinct autoprocessed forms—p20/p12, p22/p10, and p20/p10. While both p22/p10 and p20/p10 forms of caspase-4 are capable of processing GSDMD, how these GSDMD-cleaving states are structurally organized in complex with full-length human GSDMD remains unclear. Here, we present cryo-EM structures of full-length human GSDMD bound to two human caspase-4 autoprocessed forms, p22/p10 and p20/p10. Both complexes preserve exosite-mediated recognition of the GSDMD C-terminal domain, but they display distinct catalytic-groove occupancy. In the p22/p10 complex, a residual LEED-containing IDL segment folds back into the catalytic pocket, whereas in the p20/p10 complex, the GSDMD FLTD cleavage-site linker occupies the same groove. These structures reveal how distinct IDL-processing states are associated with different modes of catalytic-groove occupancy and provide a structural framework for understanding full-length GSDMD recognition by human caspase-4.