Modulating effects of the plug, helix, and N- and C-terminal domains on channel properties of the PapC usher.

The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane beta-barrel channel, an internal plug domain that occludes the channel, and globular N- and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an alpha-helix that caps the plug domain, or the N- and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and alpha-helix function to modulate the gating activity of the PapC twin pore.