Extended Data Fig. 10: The CRID3-binding site in NLRP3.
From: Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3
a, CRID3 binds into a deep crevice that is spanned by subdomains NBD, HD1, WHD, HD2 and trLRR. Only the tertiary alcohol group reaches out of this binding cleft. b, Close-up of CRID binding to subdomains NBD, HD1 and WHD (left) and HD1, WHD, HD2 and trLRR (right). To visualize the binding sites, the other subdomains in each case were omitted. c, Density map around CRID3 displayed at 4 (black), 7 (red) and 10 (magenta) RMSD threshold. d, SPR measurements of NLRP3 mutants A228Q, R351T and R578A showed no binding to CRID3. e, Mutational analysis of the CRID3-binding interface in NLRP3 shows that all three mutants could not be activated by nigericin in cell-based assays. This suggests that the integrity of the binding site in between the NBD/HD1 and WHD/HD2 subdomains is critical for the activation mechanism of NLRP3. An ASC speck activation assay was used with HEK293T cells stably expressing an ASC-BFP fusion and transfected with a doxycycline-inducible NLRP3-T2A-mCherry construct containing either wild-type (wt) or CRID3-binding interface mutants. Data are mean ± SEM of n = 3 independent experiments (ns p > 0.05, * p < 0.05, **** p < 0.0001) (two-way ANOVA with Tukey’s multiple comparisons test).
