Combined structural and FMO-based insights into shaft pilin polymerization mechanism in Streptococcus sanguinis

Bacterial pili are proteinaceous polymers that facilitate diverse biological functions. The SK36 strain of oral commensal bacterium Streptococcus sanguinis harbors sortase-assembled pili consisting of four proteins; PilA, PilB, PilC, and PilX. However, details regarding their structures and assembly mechanisms remain unclear. The crystal structures of recombinant PilA and PilB backbone pilins were examined at resolutions of 3.2 Å and 1.8 Å, respectively. Both exhibit a three-domain architecture (domains 1-3 from N- to C- terminus) with intramolecular isopeptide bonds in domains 2 and 3, and a positively charged, highly hydrophobic cleft in domain 1. Notably, while alignment along the same axis within the crystal was noted, their molecular orientations differ, as PilA maintains identical orientations (linear form), whereas PilB molecules are flipped 180° relative to each other (helical form). Both recognize the conserved ALLPNT sequence of domain 3 via the domain 1 cleft. Fragment molecular orbital calculations revealed no significant energetic differences between the linear and helical forms, with interactions predominantly mediated by C-terminal asparagine and threonine residues. Immunoblot analysis confirmed intermolecular isopeptide bond formation between threonine and conserved lysine residues at the domain 1-2 interface. The preceding glycine residue in the GALLPNT sequence may serve as a flexible pivot, enabling transitions between both forms. Since PilA, PilB, and PilC contain the GALLPNT sequence and could interconnect, the observations of domain 1-mediated recognition of the domain 3 C-terminal region indicate a fundamental mechanism governing S. sanguinis pilus assembly. These findings provide molecular-based insight into sortase-mediated pilus biogenesis in Gram-positive bacteria.