Extended Data Fig. 3: Binding affinities of TcB–TcC, TcB–TcC(D651A) and empty TcB–TcC for TcA and of TcB–TcC for TcA(L2422E).
From: Tc toxin activation requires unfolding and refolding of a β-propeller
a, Interaction of TcA with TcB–TcC(WT), empty TcB–TcC, or TcB–TcCD651A, and interaction of TcA(L2422) with TcB–TcC were measured by BLI. KD, kon and koff obtained from global fits are shown. Data are mean ± error of fit; 5–7 individual curves were included in the global fits. b–e, BLI sensorgrams of TcA(WT) interacting with immobilized TcB–TcC(WT) (b), TcB–TcC(D651A) (c), empty TcB–TcC (d) and TcA(L2422E) interacting with immobilized TcB–TcC(WT) (e). TcA pentamer concentrations were 1.25 nM–80 nM in b and c, 16–1,000 nM in d and 2.8–180 nM in e. A global fit according to a 1:1 binding model was applied (black dashed curves). Resulting KD, kon and koff values are shown. Association and dissociation phases are separated by a grey dotted line. f, g, Negative-stain electron micrographs of TcA(WT) (f) and TcA(L2422E) (g) incubated with wild-type TcB–TcC. TcA (200 nM) was incubated with TcB–TcC (300 nM), and the excess of free TcB–TcC was removed by size-exclusion chromatography before imaging. Red circles in g highlight side views of TcA without TcB–TcC. Experiments were performed three times with comparable results. Scale bar, 200 nm.
