Extended Data Fig. 3: Structural and functional analyses of IpaH7.8 recognition of GSDMD.
From: Structural mechanisms for regulation of GSDMB pore-forming activity
a, Structural modeling of IpaH7.8 recognition of GSDMD. Structure of GSDMD-N domain from FL GSDMD (PDB: 6N9O) is overlaid with that of GSDMB-N domain (from the IpaH7.8LRR–GSDMB complex). The binding interface is highlighted by a red box with structural elements labeled. b, Analyses of GSDMD degradation by IpaH7.8 mutants deficient in recognizing GSDMB. Flag-IpaH7.8 (WT, C357S, or a binding-site mutant) was co-expressed with GSDMD in 293T cells. Cell lysates were subjected to immunoblotting. c, d, Alignment of the N-terminal domain sequences of human gasdermins and mouse GSDMD. Second structural elements of GSDMB-N domain are numbered and shown on top of the sequences. Numbers of starting residues are indicated on the left. Identical residues are in red background and conserved ones are in red. Residues involved in binding IpaH7.8 are highlighted in cyan (c) or blue background (d). e, Functional validation of IpaH7.8 mutants in blocking GSDMD-mediated pyroptosis. Flag-IpaH7.8 (WT or an indicated mutant) and GSDMD were co-transfected into 293T cells and active CASP4 p20/p10 proteins were electroporated into the cells to induce GSDMD-dependent pyroptosis. IpaH7.8-induced degradation and CASP4 cleavage of GSDMD were examined by immunoblotting. SDS-PAGE analyses show the loading of CASP4 p20/p10 proteins. LDH release-based cell death data are means (bars) of three replicates (circles). f, Assay of mouse GSDMD degradation by IpaH7.8. Flag-IpaH7.8 (WT or C357S) was co-expressed in 293T cells with human or mouse GSDMD or human GSDMD with its L16−E21 sequences substituted with the mouse sequences (V16−D22). Cell lysates were subjected to immunoblotting. Data (b, e, f) are representative of three independent experiments. See Supplementary Fig. 1 for gel source data.
