Supplementary Figure 6: Mutagenesis verifies the binding mode of the C₂B–SNARE complex.

(a,b) Plots of normalized intensities of the SMRs in 1D ¹³C-edited ¹H-NMR spectra of 3 μM WT or mutant ¹³C-C₂AB as a function of SNARE complex concentration. C₂AB mutants contained single substitutions in basic residues as indicated and color-coded. The data were acquired in 25 mM Tris (pH 7.4), 125 mM NaCl and 1 mM CaCl₂, and were fitted to a single-site binding model². (c,d) Bar diagrams illustrating the K_ds derived from fitting the data of Figs. 5b,c to single-site binding models. The K_d values should be interpreted with caution because we did not use SNARE complex concentrations beyond 20 μM to minimize contributions from weaker binding modes and, as a consequence, the titrations were far from reaching saturation for the mutants with stronger effects on binding. Because of the uncertainty in the limiting intensities at infinite SNARE complex concentrations for the mutants that bind to the SNARE complex more weakly, this limiting value was forced to be 0.555 times the intensity at 0 SNARE complex concentration. The 0.555 factor was derived from averaging the ratios between intensities at 0 and infinite SNARE complex concentration in the fits obtained for WT C₂AB. Bars show average K_ds calculated from two independent experiments, and error bars show standard deviations.