Extended Data Fig. 11: The binding mechanism of ICCB-19/Apt-1 on TRADD.

a-c, Superposition of 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled His-TRADD-N (250 μM) in the presence (red) and absence (blue) of Apt-1 (500 μM) (a), ICCB-19 (500 μM) (b) or ICCB-19i (500 μM) (c). The close-up view of the region exhibited large perturbations was shown right. d, Binding pose of ICCB-19 in complex with TRADD-N was generated by induced-fit docking. The left panel demonstrated the shape and polarity of the ligand binding pocket surface, with red regions indicating negatively charged and blue positively charged. The right panel showed details of the interactions between the compound and TRADD-N. The compound was shown as cyan sticks, and the protein was shown as pink cartoon with key residues highlighted in sticks. Hydrogen bonds were shown as red dashed lines. e, Coomassie blue staining of WT and each mutant protein for thermal shift assay. f, g, HEK293T cells were seeded at 7.5 × 10³ cells per well in a white, clear-bottom 96-well plate 24 h before transfection. Cells were then transfected with the indicated plasmids for 24h. Medium was removed and replaced with Opti-MEM medium (100 μl) for 1 h at 37 °C. The Nano-Glo reagent was prepared and added to each well immediately before the luminescence reading was taken. Luminescence was measured immediately on a plate reader and reported as relative light units (RLU). Mean ± s.d. of n = 6 biologically independent samples, representative of 3 independent experiments.

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