Homotypic CARD-CARD interaction is critical for the activation of NLRP1 inflammasome

摘要

a Size-exclusion chromatograph of the MBP-ASCCARD/NLRP1CARD complex. ASCCARD was fused with an N-terminal His-tag and incubated with untagged NLRP1CARD, which was first purified by Ni-affinity chromatography. The complex eluted in the void position on a SuperdexTM 24 gel filtration column. b A negative-stain EM image of MBP-ASCCARD/NLRP1CARD complex. c Size-exclusion chromatograph of the MBP-ASCCARD/NLRP1CARD complex in different salt concentrations. d High salt significantly disrupted filament formation. e Y2H analysis of the NLRP1CARD and ASCCARD interaction. Yeast cells co-expressing GAL4 DNA-binding domain (BD)-ASC CARD fusion and GAL4 activation domain (AD)-NLRP1CARD fusion were grown on agar plates lacking leucine and tryptophan (-Leu/-Trp) for transformant growth and lacking histidine, leucine, and tryptophan (-His/-Leu/-Trp) for detecting CARD–CARD interaction. Three individual clones for each combination were plated. (–) denotes empty vector control. f Measurement of the protein–protein interaction of wild-type NLRP1CARD with ASCCARD by the M2H experiment. Luciferase activity in the HEK293T cells was normalized to Renilla and data were presented as the fold of negative control. Mean values ± SEM are representative of three independent experiments. g Structure-based sequence alignment of NLRP1CARD ({"type":"entrez-protein","attrs":{"text":"NP_127497.1","term_id":"14719829","term_text":"NP_127497.1"}}NP_127497.1) and ASCCARD ({"type":"entrez-protein","attrs":{"text":"NP_037390.2","term_id":"10835256","term_text":"NP_037390.2"}}NP_037390.2). Different colors are highlighted to show the interfacial residues involved in the three asymmetric interactions of death domain superfamily. The secondary structure of NLRP1CARD and ASCCARD are labeled on the top and bottom, respectively.