Fig. 3

Identification of HBEGF amino acids critical for diphtheria toxin (DT)-mediated cytotoxicity through PASTMUS. a Identification of HBEGF-targeting sgRNAs conferring cell resistance to DT. Distribution of sgRNAs mapped to the corresponding amino acid in HBEGF is indicated on top. b a.a. deletion fold change corresponding to each amino acid. The solid blue bars indicate single-a.a. deletions; bars with transparency indicate multiple-a.a. deletions. The width of the blue bar with transparency corresponds with a.a. deletion length. c a.a. combo mutation fold change corresponding to each a.a. Width of the bar indicates affected a.a. length. d Schematic diagram of HBEGF with the EGF-like domain shown in green, a known binding region for DT. e Essential score of each a.a. of HBEGF. Cutoff of essential score is plotted as a dashed line, with critical amino acids above the cutoff shown in black and known critical amino acids labeled in red. f Effects of single-a.a. deletions on the susceptibility of cells to DT. Cells were treated with different concentrations of DT, and the MTT cytotoxicity assay was performed 48 h after toxin treatment. Data are presented as the mean ± SD, n = 5. g Surface diagram of crystal structure of the complex of DT with EGF-like domain of HBEGF. The EGF-like domain of HBEGF is shown in gray, and the receptor-binding domain of DT is shown in cyan (PDB code: 1XDT)