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 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.
Highlights
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)
The following regions were deleted either alone or in combination (Fig. 1B): the N- and C-terminal domains (⌬N⌬C), the plug domain (⌬plug), the 5– 6 hairpin that interacts with the plug (⌬hairpin), and the ␣-helix that caps the hairpin (⌬helix)
Each of the PapC mutants was compared with the wild type protein for expression level in the OM and resistance to denaturation by SDS, which provides an indication of proper folding and stability of the -barrel domain (26)
Summary
Media and Reagents—Dodecyl-maltopyranoside and lauryl(dimethyl)amine oxide were purchased from Anatrace, and N-octyl-oligo-oxyethylene was from Axxora. Fractions containing PapC were pooled, and the His tag was cleaved off PapC by overnight digestion at room temperature with 1.5 units of thrombin/mg of PapC while dialyzing against 20 mM Tris-HCl (pH 8) and 0.12 M NaCl. The thrombin was inhibited by addition of phenylmethylsulfonyl fluoride, and the mixture was subjected to a second round of nickel affinity chromatography in buffer A. Proper folding of the PapC constructs in the OM was checked by resistance to denaturation by SDS, as determined by heat-modifiable mobility on SDS-PAGE These procedures were performed as described (20, 22). An aliquot of pure protein was added to one chamber only (cis-side), from a 5–100-fold dilution of the purified protein (0.3- 0.6 mg/ml) in a 1% N-octyl-oligo-oxyethylene solution in the 1 M KCl buffer. The data were stored on a PC computer using the Acquire software (Bruxton) and analyzed with pClamp
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