Fig. 4. cFLIPtDED self-assembly is crucial for the formation of the CFH7G-C8FGLG complex.
a Illustration of five representative interfaces involved in the cFLIP-cFLIP interaction in the CFH7G-C8FGLG complex. Interface residues and the cFLIP mutations generated in this study are depicted. Thick green lines highlight the type III-II-III interface, while thick blue lines highlight the representative interfaces between two type III-II-III CSS-mediated cFLIPtDED trimers. Residues used in mutagenesis studies are highlighted with red boxes, while glycine residues are indicated by red dots. ‘Lp’ denotes loop regions. The list in the black box outlines the positions of CF mutations on the surfaces displayed in (a) and (b). The molecules are colored as their counterparts in Fig. 3g. b Illustration of five interfaces involved in the interaction between the cFLIP double-layer intermediate complex and Casp-8 within the same complex. Thick red lines highlight the Casp-8-recruiting CBS on the cFLIP double-layer intermediate complex. Interface residues and the cFLIP mutations generated in this study are depicted. c Pulldown assay of CFH7G and mutant CFH7G by His-tagged C8FGLG demonstrating the importance of cFLIPtDED self-assembly in the formation of the binary complex. Resin-bound fractions are divided into the eluted protein fractions and the resin after elution fractions, each analyzed by SDS-PAGE to assess the amount of bound CF and His-tagged C8FGLG. Protein bands were quantified using ImageJ version 1.50i (https://imagej.net/ij/). The bar chart made by Excel version 16.54 shows the quantified mutagenesis results of (c) (therefore, n = 1), with the ratios of cFLIP to Casp-8 plotted as blue and light blue bars for the eluted protein and resin fractions after elution, respectively. Each ratio is normalized to that of lane 14, with normalized results plotted as orange and yellow bars, respectively, and the ratio indicated on top. “Type a” and “type b” cFLIP mutations are indicated. d SDS-PAGE analysis result of the flow through fractions from (c). The experiments of (c) and (d) were repeated twice with similar results. Source data are provided as a Source Data file.