Fig. 3: Interaction of WTX and its mutants with lipid vesicles.
a Amide-aromatic regions of 1D NMR spectra of WTX measured at different vesicle concentrations. The drop in intensity of NMR signals corresponds to binding of the toxin to the vesicles and decrease in the apparent fraction of the free toxin in solution (Pfree*). b Relative attenuation of intensities of backbone NH cross-peaks in the 15N-HSQC spectrum of WTX induced by addition of POPC/POPG/CHOL vesicles. Data are values obtained from a single NMR experiment. Error bars are experimental errors. The 0.5 threshold line subdivides the data in two groups: the residues interacting with the vesicles or not. Color code corresponds to the regions highlighted by ellipses on c. c Spatial structure of WTX[P33A] mutant (used for the model building, see Methods) in ribbon representation. Disulfide bonds, positively charged, and negatively charged residues are colored in orange, blue, and red, respectively. The protein ribbon is colored according to vesicle-binding data from b. The residues interacting with the membrane are in green. Two protein regions responsible for protein-membrane interaction are shown by dashed ellipses. d Binding isotherms for WTX and its mutants are approximated by the Langmuir equation. Fitted parameters are summarized in Supplementary Table 3. The Pfree values shown are the mean ± S.E.M. of three independent titration experiments (n = 3). *p < 0.05 and ***p < 0.001 indicate the significant difference between groups by Repeated-Measures one-way ANOVA/Tukey’s test. Please note that obtained isotherms represent upper approximation to the real binding parameters. In the presence of slow-intermediate (on NMR timescale) exchange between the free toxin in solution and membrane bound toxin, the apparent Pfree* value calculated from the NMR signals intensity is always lower than true Pfree value.
