Fig. 1: Structure of the KlebC–TolC-binding domain and its binding to KqTolC.

a Cartoon representation of the KlebC_51-254 TolC-binding domain crystal structure. Leu86, Ala107, Tyr177 and Leu198, mutated to form disulphide bonds are shown as yellow spheres. Side chains of FRET pair Trp81 (green sticks) and Gln204 (red sticks), which was mutated to Cys, are also shown. b ITC titration of 84 µM KlebC_1-254 into 7.6 µM KqTolC (filled squares) and 89 µM KlebC_51-254 into 5.9 µM KqTolC (offset by 0.06 µcal s⁻¹ in top panel and open circles in bottom panel). Data were fitted to a one set of sites fit to give K_d = 35 ± 7 nM, N = 0.90 ± 0.03 binding sites per TolC trimer and ΔH 30.6 ± 2.6 kcal mol⁻¹ and K_d = 368 ± 16 nM, N = 1.34 ± 0.12 binding sites per TolC trimer and ΔH 20.8 ± 1.0 kcal mol⁻¹ for KlebC_1-254 and KlebC_51-254, respectively. Typical traces are shown, values are averages of duplicate experiments. c Pre-equilibrium fluorescence increase in tryptophan emission upon complex formation between 0.5 µM KqTolC and 7.5 µM KlebC_1-254. Single exponential fit to the data to determine k_app is shown in red. Inset, Dependence of k_app on KlebC_1-254 concentration. Data are averages of duplicate experiments fitted to a straight line, the gradient of which gives the association rate constant, 1.9 ± 0.1 × 10³ M⁻¹ s⁻¹. d Liquid growth curves of K. quasipneumoniae Qmp M1-977, with the addition of KlebC-E9 DNase (black), A107C, Y177C KlebC-E9 DNase (reduced, solid red; oxidised, dashed red), L86C, L198C KlebC-E9 DNase (reduced, solid blue; oxidised, dashed blue), or no KlebC-E9 DNase (green), added after 60 min. Data shown are from one representative experiment out of three biological repeats. e In total, 200 µl KqTolC (green), KqTolC + L86C, L198C KlebC_1-254 (reduced, solid blue; oxidised, dashed blue), and KqTolC + A107C, Y177C KlebC_1-254 (reduced, solid red; oxidised, dashed red) were loaded onto a 10/300 S200 column. f Fluorescence emission of 1 µM Q204C^AEDANS KlebC_1-254 in the absence (green) and presence (red) of 1 µM KqTolC. λ_Ex = 280 nm, slit widths = 2 nm.