Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

Abstract

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 ␤-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 ␣-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 ␣-helix function to modulate the gating activity of the PapC twin pore.The cell envelope of Gram-negative bacteria contains a vast array of protein machineries dedicated to the translocation of polypeptides across the cytoplasmic membrane, periplasm, and outer membrane (OM) 3 (1, 2). Some of these complexes also participate in the assembly of surface-exposed appendages, such as flagella and pili (fimbriae). One of the most thoroughly studied secretion systems is the chaperone/usher pathway, responsible for the biogenesis of a superfamily of virulenceassociated surface structures, including P and type 1 pili (3). These pili play essential roles in the pathogenesis of uropathogenic Escherichia coli by providing a tool for attachment of the bacteria to host urothelial cells (4 -6). P pili, encoded by the chromosomal pap gene cluster, are critical virulence factors for infection of the kidney by uropathogenic E. coli and the development of pyelonephritis. The P pilus is composed of multiple subunits of PapA, which form a rigid helical rod. A thin linear tip fibrillum is located at the distal end of the pilus and is made of four different subunits (PapK, PapF, PapE, and the adhesin PapG) that assemble in a precise order and stoichiometry (3). The minor pilin, PapH, anchors the pilus rod to the cell surface (7).The pilus subunits are first translocated through the cytoplasmic membrane via the Sec general secretory pathway (8). Once in the periplasm, the subunits form binary complexes with the PapD chaperone. The details of the binding interact...

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis

“…In the apo usher, in the absence of a polymerizing and/or anchored pilus, the β-sandwich PLUG occupies the inactive pore. The PLUG seems to be kept stable in the pore via its interactions with residues of the β-barrel interior wall as well as the β5-6-hairpin loop and an α-helix of the TD that caps the β5-6-hairpin loop (3,25). In the active form, as seen in the FimDCH structure, the FimD pore rearranges to a nearly circular pore of 32 Å diameter (5,24) and the PLUG is relocated to the periplasm, where it forms a high-affinity, stable interaction with the NTD (23).…”

Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

“…Mutations of the NTD or either CTD abolish pilus biogenesis (25,27). The Plug domain is also required for pilus biogenesis (29,30). A Plug-deleted PapC folds but does not assemble pili, suggesting that the Plug domain plays a direct role in catalyzing pilus biogenesis (29,30).…”

“…The Plug domain is also required for pilus biogenesis (29,30). A Plug-deleted PapC folds but does not assemble pili, suggesting that the Plug domain plays a direct role in catalyzing pilus biogenesis (29,30). Binding studies have suggested that the chaperone-adhesin complex is initially targeted to the NTD of PapC (26), and this is thought to activate the usher protein such that the β-sandwich Plug domain shifts from the channel, where it is located in the apo usher, to the periplasmic space, resulting in an open translocation pore (28).…”

Molecular mechanism of bacterial type 1 and P pili assembly

“…One major step in usher activation is the swinging out of the plug from its position within the usher pore lumen to its position in the periplasm where it interacts with the usher NTD. Electrophysiological studies suggested that both the NTD and the CTDs are involved in gating the usher, even in the absence of a translocation substrate [61], with only the FimH adhesin being able to stabilize the plug-NTD interaction in the open state while reaching out for the pore [43,47] ( figure 4, view b). Recently, it was proposed that the lectin domain of FimH actively displaces the plug domain, because the plug domain was calculated to have weaker interactions with the usher lumen than the lectin domain of FimH [27].…”

“…Recently, it was proposed that the lectin domain of FimH actively displaces the plug domain, because the plug domain was calculated to have weaker interactions with the usher lumen than the lectin domain of FimH [27]. Deletion of the plug domain showed that it is essential in pilus biogenesis [61,62], its role in active subunit recruitment becoming recently apparent: the NTD of PapC alone was only able to bind PapD-PapG and PapD-PapE, but not the other chaperone-subunit complexes PapD-PapK/A/H, whereas an NTD-plug complex is able to bind all chaperone-subunit complexes [47]. ( figure 4, view c).…”