Fig. 5: Importance of the cysteine residue of P. gingivalis PorG and structure of the PorKN Hub complex.

a, b P. gingivalis PorG Cys232 is required for T9SS activity. Similar results were obtained from 3 biological repeats. wt, wild type. porG^ALFA, codes for PorG with an ALFA-tag epitope. a Strains containing the indicated porG alleles were grown on blood agar. b Equivalent strains constructed in a gingipain-deficient background were analysed by whole cell immunoblotting. c CryoEM volume for a 33-mer P. gingivalis PorKN complex at 4.0 Å resolution viewed from the side and from the periplasm. The inset shows the structure of the repeating PorKN unit obtained at higher (2.4 Å) resolution. The structure is depicted at a high contour level (solid representation with a single PorKN unit shown in color) and at a low contour level (transparent envelope) to visualize the detergent micelle, which approximates the position of the OM. d Structures of PorK and PorN in cartoon representation and rainbows colored from N- (blue) to C-terminus (red), viewed from the inner rim of the PorKN ring and with the OM at the Top. The bound Ca²⁺ ion is shown in atomic sphere representation. Glycosylated serine and threonine residues are labelled. e The PorKN structure from c (colored density) placed into the cryoET volume of the complex resolved previously in intact cells³⁴ (EMD-24227). See also Supplementary Movie 5. f View of the PorKN heterodimer unit in cartoons representation, OM at top, illustrating the triangular cross-section of the PorKN ring. g Space-filling model of a PorKN pair and its immediately neighboring pairs viewed from the OM (Top) and from the outer rim of the PorKN ring with the OM above (Bottom). The position of the PorG-reactive Cys 356 on PorK is shown (yellow). See also Supplementary Movie 6. h Schematic summarizing the areas buried by the different inter-subunit interfaces of the PorKN complex. Protomers are oriented as in (d). i Structure of the external loop bearing the PorG-reactive cysteine 356 of PorK with density for the motif overlaid.