Figure 1.
Sequence comparison of the α-hairpins with the anti-parallel coiled-coil structures.
Sequences of the RLS (or RLT) motif from six adaptor proteins are aligned. The corresponding heptad positions are shown above the sequence. The three conserved residues in the RLS (or RLT) motif are indicated by arrows. The12 RLS (or RLT) motif residues are in a box, which was used in the MacA-HlyD12 hybrid. The 18 and 24 residues used in the construction of MacA-HlyD18 and MacA-HlyD24, respectively, are indicated (D18 and D24). The amino acid numbering is based on the protein precursors. In the sequence alignment, Ec, Pa, Aa, and Mh stand for E. coli, P. aeruginosa, Actinobacillus actinomycetemcomitans, and Mannheimia haemolytica, respectively.
Figure 2.
HlyA activity of E. coli SE5000 expressing wild-type or mutant HlyD.
A. Hemolytic activity of E. coli cells expressing wild-type or mutant HlyD. The absorbance at 450 and 543 nm was detected using a spectrophotometer, and hemolytic activities were calculated according to the following formula: Percent Hemolysis = (1-ODs/ODt)×100, where ODs and ODt are the differences in optical density at 543 nm between the samples and 100% hemolyzed SRBC solution, and differences in optical density at 450 nm between nonhemolyzed (control) and 100% hemolyzed sheep red blood cell solution, respectively. Filled symbols and lines indicate hemolytic activity detected in culture supernatants (OD543/OD450). B. Effects of expression of HlyD mutants (HlyD-R131A, HlyD-L186A, HlyD-T190A, HlyD-T197Y, HlyD-null) on HlyA secretion. E. coli SE5000 coexpressing HlyA, HlyB, HlyC, and wild-type or HlyD mutants (HlyD-R186A, HlyD-L190A, HlyD-T197A, HlyD-T197Y, or HlyD-Stop) were grown exponentially in LB with 10 mM CaCl2 at 37°C, and proteins in the culture supernatant (equivalent to 3.5 OD600 of cells) were TCA precipitated. The precipitated protein samples were separated by SDS-PAGE and subjected to immunoblotting with HlyA antibody (upper panel). Total cell lysate was used for the western blot analysis of HlyA to detect its expression levels in the cell (lower panel).
Table 1.
Degree of hemolysis by E. coli cells expressing HlyD mutants.
Figure 3.
Interaction between TolC and HlyD in vivo.
The in vivo interaction between HlyD and TolC was detected using a chemical cross-linking agent (DSP). E. coli BW25113ΔacrAB ΔtolC210::Tn10 cultures that coexpressed HlyA, HlyB, HlyC, hexa-His-tagged wild-type TolC, and c-Myc-tagged wild-type HlyD (WT) or one of the HlyD mutants (R186A, L190A, T197A and T197Y) are shown. All cultures were treated with (+) or without (−) DSP. Affinity-purified TolC and cross-linked proteins (HlyA and HlyD) were separated by SDS-PAGE and immunoblotted using monoclonal antibodies to His-tag and c-Myc, and polyclonal antibodies to HlyA.
Figure 4.
Interaction between the HlyD RLT motif and the TolC α-barrel tip region.
A. The MacA-TolCα hybrid (T) was coupled to the CNBr-activated resin or the inactivated resin by Tris and incubated with a MacA-HlyD hybrid protein (D24, D18, or D12; H). After washing, the resin was applied to the SDS-PAGE gel. Only the MacA-HlyD12 hybrid protein was bound to the MacA-TolCα-coupled resin. The E. coli MacA (M), which was known to bind to the MacA-TolCα hybrid protein [22], was used as a positive control. B. Interaction of the E. coli MacA-HlyD hybrid (wild-type and mutants) and MacA-TolCα hybrid on a size-exclusion chromatographic column. Elution profiles of the wild-type MacA-HlyD12 hybrid and its mutants (R186A, L190A, T197A, and T197Y) were co-injected with the MacA-TolCα hybrid protein. The arrows with the molecular mass indicate the fractions corresponding to the calculated molecular size from the elution volume. The box around 560 kDa as a molecular size indicates complex formation from the two proteins. The elution profiles of MacA-HlyD12 hybrid proteins alone are shown in Figure S1. C. In vitro binding assay to confirm the results of the size-exclusion chromatography. The same proteins were used as in (B), and the experimental methods were used as in (A). Results were similar to those produced in (B), except for the augmented affinity between the MacA-HlyD12 hybrid T142A mutant and the MacA-TolCα hybrid.