Fig. 1

Truncation of L5 affects folding of the Pet passenger domain in bacterial cells. Topology model of the Pet β-barrel domain (a) based on the crystal structure of the EspP β-barrel domain (PDB code 3SLJ) (these β-barrels share ~ 91% sequence identity) (b). The structure shows L5 in an ‘open’ conformation where it interacts with L4, forming a 4-stranded β-sheet. It is noteworthy that the uncleaved passenger domain that traverses the length of the pore is hidden for clarity. For a, yellow and white squares indicate residues with side chains that point toward the lipid bilayer and the β-barrel lumen, respectively. Residues in periplasmic turns labeled T1–T5 and extracellular loops labeled L1, L2, and L6 are shown in gray, as are the residues in the α-helical segment. Residues in extracellular loops labeled L3, L4, and L5 are shown in green. Given that these loops are mobile, the alignment of L4 and L5 does not strictly follow PDB 3SLJ. c The domain structure (left) and schematic for biogenesis (right) show that, post cytoplasmic synthesis and inner membrane translocation, Pet is processed by a signal peptidase to a ~ 136 kDa polypeptide in the periplasm. The β-barrel domain of Pet is then assembled into the outer membrane to allow translocation of the passenger domain, in a C- to- N- terminal direction, until the extreme N-terminus traverses the β-barrel pore. An autocatalytic cleavage reaction within an intra-barrel α-helical segment liberates the ~ 106 kDa mature form of the passenger domain, leaving the ~ 30 kDa β-barrel domain embedded in the outer membrane. d Topology model of the Pet β-barrel domain showing the L5 truncation created by replacing E₁₂₃₃–K₁₂₄₆ with one glycine residue (shown in red). Pulse-chase expression of Pet and PetΔL5 and sensitivity to proteinase K (PK) in E. coli TOP10 (e) and BW25113ΔompT (f) monitored by SDS-PAGE and immunoblotting with anti-Pet passenger domain antibodies. All samples were TCA precipitated prior to SDS-PAGE. g Heating of total membranes containing Pet and PetΔL5 at the temperatures indicated. Samples were analyzed by SDS-PAGE and immunoblotting with anti-β-barrel domain antibodies. Images are representative of at least two independent experiments