Extended Data Fig. 2: Binding between FtsN and FtsA is dominated by electrostatic interactions.
a. Binding curves obtained from the MST thermograms of Alexa Fluor 647 FtsA titrated with full length FtsN at 100 (black symbols), 150 (green symbols) or 300 mM KCl (red symbols). The binding curves at the lower ionic strengths are biphasic with a first transition from 100 to 1000 nM FtsN and a second transition that could not be saturated. The interaction was significantly weaker in the presence of 300 mM KCl (red symbols). The Hill equation, with n fixed to 1 was used to calculate the midpoint of the first transition for the curves at 100 mM (black line) or 150 mM KCl (green line), resulting in apparent KD values of 250 ± 51 nM at 100 mM KCl or 734 ± 23 nM at 150 mM KCl, n = 3. b. The mutant peptides D5N (n = 5), RAAK (n = 5), D5N-RAAK (n = 3) efficiently colocalized with the FtsZ filaments similar to the wildtype peptide, while FtsNcytoDDEEHis (n = 5) mutant and the scrambled FtsN peptide (n = 6) show only weak colocalization Scale bars are 5 µm. c. Sequences of tested peptides. Dashed boxes correspond to the conserved D5 and the RRKK motifs, which are lost in the scrambled peptide. d. The colocalization efficiency of the scrambled peptide (n = 7, 0.24 ± 0.1, mean ± s.d.) was significantly decreased in comparison to FtsNcytoHis (n = 5, 0.62 ± 0.1) and FtsNcytoD5N-RAAKHis (n = 3, 0.55 ± 0.06) (P value = 1.15 × 10-4 and 1.19 × 10-3 respectively). Each dot corresponds to an independent experiment acquired within the same batch of proteins. The boxes indicate the 25–75th percentiles, whiskers the outliers, the midline indicates the median and square indicates the mean. P values were calculated using a two-tailed Student’s t-test for parametric distributions. e. Structure of the FtsA dimer in complex with the C-terminal peptide of FtsZ46. The surface charge of one FtsA monomer is shown. The 1 C domain (dashed square) contains a large negatively charged patch, which is oriented towards the membrane surface, where it can interact directly with positively charged FtsN peptide.
