Figure 4. Mapping of residues essential for pilus assembly and hypervariable regions on the PpdD pilus surface.

(A) Surface electrostatic potential of the PpdD pilus (B) Surface representation of the PpdD pilus structure showing the distribution of charged residues essential for pilus assembly (positively charged in blue and negatively charged in red). Non-essential charged residues are colored in green. Surface of a single PpdD subunit is outlined in black. (C) Surface representation of a PpdD pilin subunit in the pilus colored as in (A). The surface-exposed side of the subunit (front view) displays predominantly residues permissive for charge inversions. The back view (180° rotation) shows the clustering of charged residues essential for pilus assembly on the buried face of the pilin. (D) Piliation assay with single charge inversion PpdD variants. Cells (top) and sheared pilus fractions (Pili, bottom) corresponding to 0.05 OD_600nm of bacteria were separated on SDS-PAGE and analyzed by immunoblot using anti-PpdD antibodies. (E) Quantification of the fraction of PpdD assembled into pili. The bars represent the mean values and error bars represent standard deviation from 4 independent experiments. Statistical analysis was performed by using ANOVA test to compare datasets from native PpdD and its variants: ns: non-significant difference; **: p<0.01; ****: p<0.0001. (F) Sphere representation of the PpdD pilus structure where the location of hypervariable regions is highlighted (α/β loop in green, β3/β4 loop in cyan and D-region in magenta). A single PpdD subunit is outlined in black. (G) Schematic representation of the topological arrangement of neighboring subunits in the PpdD pilus structure. The location of hypervariable regions is indicated and colored as in (F). The size of the buried surface area in each inter-protomer interface is proportional to the width of the grey line.