Fig. 4: Interfaces in the open octamer are critical in the NLRP3 activation and assembly.

A Mutations at the interfaces did not impair NLRP3 expression level. The component proteins were detected using Western blots. These experiments were repeated twice. B Cellular assay showing induction of IL-1β signaling upon transfection with 2.5, 5, or 10 ng of WT or mutant FL NLRP3 vectors. EV, empty vector control. Bars indicate the mean, and the data points from six independent experiments are shown. C Structural and sequence alignments around the catalytic surface around Asp₃₆₃ (top) and the Loop_504-517 region (lower), respectively. Closed (PDB ID: 7LFH), open (this study), activated (PDB ID: 8EJ4) NLRP3, NLRC4 (PDB ID: 8FW2), and NAIP (PDB ID: 8FVU) were shown as cartoon and in cyan, slate, light green, pink, and orange, respectively. The adjacent activated NLRP3 protomer was colored in dark green. Side chains of selected residues were shown in sticks in the structural alignments and highlighted with filled gray boxes in the sequence alignments. D Fluorescent mCherry measurements in HEK293TASC-mCherry cells transfected with empty vector (EV), NLRP3 WT, or mutants, with/without Nigericin treatment. Two cell images with scale bars were shown as reference. Bars indicate the mean, and the data points from three independent experiments are shown. Source data are provided as a Source Data file.