Extended Data Fig. 10: ICCB-19/Apt-1 bind to TRADD-N.
From: Modulating TRADD to restore cellular homeostasis and inhibit apoptosis
A fluorescence-based thermal shift assay was developed to quantify ICCB-19/Apt-1 binding to TRADD by measuring changes in thermal denaturation temperature (Tm). a, CBB staining of GST-tag and GST-TRADD purified from HEK293T cells. b, in vitro binding of GST-TRADD (50 μM) with ICCB-19 (250 μM) and Apt-1 (250 μM) was determined by thermal shift assay. Thermal unfolding of GST-TRADD is monitored using SYPRO Orange. Data were collected in the presence of ICCB-19 and Apt-1, leading to a rightward shift in the unfolding transition. The apparent melting temperature (Tm) is the peak in the derivative of the unfolding curve (dF/dT), which is used as an indicator of thermal stability. c, GST-tag (50 μM) does not bind to the compounds (250 μM) as determined by thermal shift assay. d, ICCB-19i does not bind to GST-TRADD as determined by thermal shift assay. e, GST-TRADD-C (50 μM) alone does not bind to either ICCB-19 (250 μM) or Apt-1 (250 μM) as determined by thermal shift assay. f-h, TRADD-N/ICCB-19 (f), TRADD-N/Apt-1 (g), and TRADD-N/ICCB-19i (h) samples used for STD NMR experiments were prepared as 1 mM ICCB-19 (f), 1 mM Apt-1 (g), 1 mM ICCB-19i (h), and 13 μM TRADD-N in 0.5 mL of PBS in D2O (10%). The on-resonance irradiation of TRADD-N was performed at a chemical shift of -0.5 ppm, whereas the off-resonance irradiation was conducted at 37 ppm. Spectra were acquired using the following parameters: spectral window of 6.4 kHz, number of scans at 320, acquisition time of 2 s, and repetition time of 3 s. The decrease in signal intensity in STD spectrum, resulting from the transfer of saturation from the protein to the ligand, is evaluated by subtracting the on-resonance spectrum from the off-resonance spectrum. This subtraction yields a positive signal from a bound ligand. The asterisks indicate the signals of the compounds. The STD data suggest that both ICCB-19/Apt-1, but not ICCB-19i, bind with TRADD-N. i, CBB staining of 6 × His- and Flag-tagged TRADD for SPR purified from HEK293T cells. The proteins were pulled down by anti-Flag affinity gel and eluted by 3 × Flag peptide. The proteins were further purified by size exclusion chromatograph on a Superdex 75 column (GE Healthcare) in a buffer containing 20 mM imidazole (pH 6.6), 200 mM NaCl, 20 mM DTT. j, BIAcore SPR analysis of ICCB-19 binding to TRADD-N. The kinetic profile of ICCB-19 binding to TRADD-N is shown. A series of concentrations of ICCB-19 (ranging from 0.3125 to 10 μM) was used to measure the binding kinetics, with TRADD-N immobilized on the CM5 chip.
