title: Electrostatic networks control plug stabilization in the PapC usher.
abstract: The PapC usher, a β-barrel pore in the outer membrane of uropathogenic Escherichia coli, is used for assembly of the P pilus, a key virulence factor in bacterial colonization of human kidney cells. Each PapC protein is composed of a 24-stranded β-barrel channel, flanked by N- and C-terminal globular domains protruding into the periplasm, and occluded by a plug domain (PD). The PD is displaced from the channel towards the periplasm during pilus biogenesis, but the molecular mechanism for PD displacement remains unclear. Two structural features within the β-barrel, an α-helix and β5-6 hairpin loop, may play roles in controlling plug stabilization. Here we have tested clusters of residues at the interface of the plug, barrel, α-helix and hairpin, which participate in electrostatic networks. To assess the roles of these residues in plug stabilization, we used patch-clamp electrophysiology to compare the activity of wild-type and mutant PapC channels containing alanine substitutions at these sites. Mutations interrupting each of two salt bridge networks were relatively ineffective in disrupting plug stabilization. However, mutation of two pairs of arginines located at the inner and the outer surfaces of the PD resulted in an enhanced propensity for plug displacement. One arginine pair involved in a repulsive interaction between the linkers that tether the plug to the β-barrel was particularly sensitive to mutation. These results suggest that plug displacement, which is necessary for pilus assembly and translocation, may require a weakening of key electrostatic interactions between the plug linkers, and the plug and the α-helix.
authors.affiliation: a Department of Biology and Biochemistry , University of Houston , Houston , TX and.
authors.forename: Thieng
authors.initials: T
authors.lastname: Pham
authors.affiliation: b Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, Stony Brook University , Stony Brook , NY , USA.
authors.forename: Nadine S
authors.initials: NS
authors.lastname: Henderson
authors.affiliation: b Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, Stony Brook University , Stony Brook , NY , USA.
authors.forename: Glenn T
authors.initials: GT
authors.lastname: Werneburg
authors.affiliation: b Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, Stony Brook University , Stony Brook , NY , USA.
authors.forename: David G
authors.initials: DG
authors.lastname: Thanassi
authors.affiliation: a Department of Biology and Biochemistry , University of Houston , Houston , TX and.
authors.forename: Anne H
authors.initials: AH
authors.lastname: Delcour
mesh_terms: D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D055672:Static Electricity
chemical_list: D029968:Escherichia coli Proteins; D018272:Porins; C111243:atpG protein, E coli
keywords: E. coli; patch clamp; secretion