Fig. 3: Characterizing TFPI-TcdB4 interactions.
a Schematic diagrams of TcdB4.2, TcdB4.21286–1805, TcdB4.21835–2367, TcdB1.1-FBD. TcdB4.1(B1.1) and TcdB1.1(B4.2) represent two mutant fragments exchanging 29 residues in the region 1432– 1600 that differ between TcdB4.1 and TcdB1.1. The numbers indicate the position of amino acid residues. GTD, glucosyltransferase domain; CPD, cysteine protease domain; DRBD, delivery/receptor-binding domain; CROPs, combined repetitive oligopeptides. Binding of 500 nM TcdB4.21286–1805 (b) or TcdB4.21835–2367 (c) to Fc-tagged TFPI and mTFPI was examined using BLI assays. Fc-tagged TFPI2, CRD2, SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. d HeLa cells transiently transfected with TFPI, TFPI2, mTFPI, SEMA6A, or FZD2 were exposed to FLAG-tagged TcdB4.21286–1805 (5 µg/mL) on ice for 60 min, washed, fixed, permeabilized, and subjected to immunostaining analysis. Expression of exogenous proteins was confirmed by detecting fused HA or 1D4 tag. Nuclei were labeled with DAPI (blue). Scale bar, 5 µm. Representative images were from one of three independent experiments. Binding of 500 nM TcdB1.1-FBD, TcdB4.21286–1805, TcdB4.2(B1.1), and TcdB1.1(B4.2) to Fc-tagged CRD2 (e) and TFPI (f) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. g Schematic diagram of TFPIβ, TFPI-K1, and TFPI-K2 fragments. Sig, signal peptide; N, N-terminal domain; K1, BPTI/Kunitz inhibitor domain 1; L1, loop 1; K2, BPTI/Kunitz inhibitor domain 2; L2, loop 2; β, GPI anchor sequence for TFPIβ. h Binding of 500 nM TcdB4.21286-1805 to Fc-tagged TFPI, TFPI-K1, and TFPI-K2 was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. i, j TcdB4.2 binding to TFPI-K2 prevents TFPI-K2 binding to its natural ligand coagulation factor Xa (FXa). FXa (0.5 ng/mL) cleaves its fluorescently labeled substrate and generates increasing fluorescent signal (measured as relative light unit, RLU, y-axis) over time (x-axis). FXa’s enzymatic activity was inhibited by TFPI-K2 (i, 7.5 ng/mL). The inhibitory effect of TFPI was blocked by adding TcdB4.21286–1805 in a dose-dependent manner (1:1, 1:3, or 1:10 molar ratio). Representative curve from one of three independent experiments is shown. FXa activity was quantified by measuring the slope of RLU curves (1–10 min) and plotted as a bar-chart (j). The same experiments were also carried out for TFPI-Fc and mTFPI-Fc, with their curves shown in Supplementary Fig. 10c, d and quantification in (j). Error bars indicate mean ± s.d.; N = 3; *, p < 0.05; **, p < 0.01 (Student’s t-test, two-sided). Source data are provided as a Source Data file.
