PMC

Model of Cai in the cytoplasmic membrane of Escherichia coli. H1, H2, H3, and H4 denote the transmembrane α-helices. The positions of cysteine residues are shown with black dots and labeled with numbers.

Model of Cai helix packing and dimerization. White and gray circles represent the four TMSs (H1–H4) of two monomers of Cai that form the dimer. Double-headed arrows indicate intramolecular disulfide bonds between Cys residues, and black lines indicate intermolecular disulfide bonds. A large gray arc represents the TMS3 helix face involved in dimerization. The dotted line indicates a potential interaction between one Cai monomer represented with white circles and another Cai monomer depicted with four hatched circles.

Cysteine residues involved in intra- and intermolecular disulfide bonds. A, aliquots of 0.2 × 10⁸ cells of the indicated strains incubated with or without Cu-OP at the indicated concentration, resolved by 15% SDS-PAGE, and probed with anti-1C11 mAb. B, EpCai_C107S/C122S cross-linking with FA. Aliquots of 0.2 × 10⁸ cells of strain producing EpCai treated (FA) or not treated (−) with formaldehyde were analyzed by 15% SDS-PAGE and immunoblotting with the anti-1C11 mAb after heating the samples at 96 °C (ΔFA) or not.

Dimer formation of mutated EpCai. For all figures, samples were subjected to 15% SDS-PAGE and probed with anti-1C11 mAb. A, expression of mutated EpCai. 0.2 × 10⁸ cells of the indicated strains were loaded on SDS-PAGE and immunodetected. B, expression of EpCai C74S at various temperatures. Strains carrying the construct encoding EpCai C74S were grown at the indicated temperature and induced for 90 min with 100 μm isopropyl 1-thio-β-d-galactopyranoside. C, influence of Cu-OP concentration on the amount of mutated EpCai dimer. See legend to A for wild-type Cai. Cells were grown at 30 °C.

Oxidized Cai is inactive. Agar plates containing or not 200 μm Cu-OP or 200 μm Cu-OP and 5 mm DTT (E) were overlaid with E. coli C600 cells constitutively producing or not producing Cai or mutated Cai (D), as indicated. Then, 1 μl of a serial dilution of ColA, ColE1, ColE2, or ColB (A), ColAAB or BBA (B–E) was dropped onto each plate. The absence of zones of killing indicates biological resistance to colicins. These experiments were performed at 30 °C.

Immunoblot analysis of cross-linked EpCai proteins containing cysteine substitutions in TMS3. A, aliquots of 0.2 × 10⁸ cells of the indicated strains were incubated with or without Cu-OP at the indicated concentration (O₂₀, 20 μm; O₅₀, 50 μm) or with 25 mm β-me (R), and analyzed by 15% SDS-PAGE and immunoblotting with anti-1C11 mAb. B, helical wheel projection of the predicted transmembrane α-helix 3 of EpCai. The percentage next to each residue is the percentage of cross-linked dimer formed after oxidation with 50 μm Cu-OP at room temperature for 20 min. The strongly interacting face of TMS3 is indicated with a solid arrow.

sp-pfColA prevents EpCai dimerization. A, strains producing EpCai, EpCai and sp-pfColA, or EpCai and sp-pfColA_B3 grown at 37 °C and incubated for 60 min with 100 μm isopropyl 1-thio-β-d-galactopyranoside (IPTG), then 30 min with 0.5 mg/ml arabinose. Samples (0.2 × 10⁸ cells) were incubated with the indicated concentration of Cu-OP and analyzed by 15% SDS-PAGE and immunoblotting with the anti-1C11 mAb (upper panel) or the anti-pfColA polyclonal Ab (lower panel). B, as in A, with strains producing EpCai L109C, EpCai V113C, EpCai Y119C, and co-producing or not sp-pfColAB3. Samples were immunoblotted with the anti-1C11 mAb only. The arrow indicates the position of the EpCai trimer. C, sp-pfColA preventing EpCai dimerization with FA. Aliquots of 0.2 × 10⁸ cells of strains producing EpCai, EpCai, and sp-pfColA were treated (FA) or not (−) with formaldehyde. Cell extracts were analyzed by 15% SDS-PAGE and immunoblotting with the anti-1C11 mAb.

EpCai forms homodimers. A, effect of Cu-OP concentration on the amount of EpCai dimer. For all the figures, samples were resolved by 15% SDS-PAGE, and anti-1C11 mAb was used for immunodetection. Aliquots of 0.2 × 10⁸ cells of the strain producing EpCai, incubated with or without one of a series of concentrations of Cu-OP, were boiled in the absence or presence of the reducing agent β-me and preincubated or not with NEM. Positions of EpCai and EpCai dimers are indicated on the right. Positions of EpCai monomers and dimers with intramolecular disulfide bond(s) are indicated as EpCai_s-s and [EpCai_s-s]₂, respectively. Molecular mass markers are indicated on the left. B, EpCai cross-linking with FA. Aliquots of 0.2 × 10⁸ cells of the strain producing EpCai were treated (FA) or not treated (−) with formaldehyde. ΔFA is similar to FA, except that samples were heated to 96 °C to break the cross-links. C, EpCai and VL1 dimer formation. Aliquots of 0.2 × 10⁸ cells of the strain producing EpCai or VL1, incubated with or without Cu-OP (400 μm), were boiled with or without β-me. Positions of EpCai, EpCai dimers, VL1, and VL1 dimers are indicated on the right. Molecular masses are indicated on the left.