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. 2011 May 20;286(20):17910-20.
doi: 10.1074/jbc.M111.238535. Epub 2011 Mar 29.

Funnel-like Hexameric Assembly of the Periplasmic Adapter Protein in the Tripartite Multidrug Efflux Pump in Gram-Negative Bacteria

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Funnel-like Hexameric Assembly of the Periplasmic Adapter Protein in the Tripartite Multidrug Efflux Pump in Gram-Negative Bacteria

Yongbin Xu et al. J Biol Chem. .
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Abstract

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.

Figures

FIGURE 1.
FIGURE 1.
AcrA hexameric structure. A, bar diagram for AcrA dimer constructs. The first AcrA unit contains the signal peptide and the lipid modification moiety to properly locate the protein at the inner membrane in the periplasm for the in vivo genetic study. For the in vitro binding assay, the signal peptide and the lipid modification moiety were removed. B, elution profiles of E. coli AcrA dimer, E. coli AcrA, and A. actinomycetemcomitans (Aa) MacA from a size-exclusion chromatographic column (Superdex 200 HR 10/30). The SDS-PAGE analyses of the fractions are shown. The numbers correspond to the fractions indicated on each chromatogram. The peaks indicated by 210, 120, and 61 (kDa) arrows are estimated as a hexamer, dimer, and monomer of AcrA or MacA protomer, respectively. C, side views of the E. coli AcrA hexamer (left) and the P. aeruginosa MexA hexamer models (right). The dimeric unit of AcrA structure (A and B chains of PDB code 2F1M), devoid of the MP domain, was used as the initial model (left). Two neighboring MexA protomers (L and M chains of PDB code 2V4D (12)), which are paired by the interaction between the MP domains, contain all four domains. The funnel stem comprising α-hairpins and the funnel mouth comprising lipoyl, β-barrel, and MP domains are indicated. D, top view of the E. coli AcrA hexameric model (left) and bottom view of the P. aeruginosa MexA hexameric model (right). The distance between MexA MP domains is indicated. Note the triangular cone-shaped internal hollows and the central pores in the modeled structures.
FIGURE 2.
FIGURE 2.
Putative pore region of the AcrA hexameric model. A, central pore in the lipoyl domains from the AcrA hexamer. Only the lipoyl domains are shown for clarity. Each protomer is colored green or cyan with transparent surface representations. The Gly-Gln-Ala sequence conserved between E. coli AcrA and P. aeruginosa MexA is shown in blue. B, alignment of the sequences around the pore-lining loops from E. coli AcrA (Ec AcrA), P. aeruginosa MexA (Pa MexA), E. coli MacA (Ec MacA), A. actinomycetemcomitans MacA (Aa MacA), and Salmonella typhimurium MacA (St MacA). The pore-lining sequences are colored in blue or magenta in a box. The conserved residues are highlighted. C, superposition of the lipoyl domains from E. coli MacA and E. coli AcrA hexamers, displayed in the Cα tracing representations. The E. coli MacA protomers are colored in gray, although E. coli AcrA protomers are in green or cyan. The pore-lining residues of MacA are in magenta, and those of AcrA are in blue.
FIGURE 3.
FIGURE 3.
Tip-to-tip interaction between TolC and AcrA. A, in vivo interaction between AcrA (L132C) and TolC (N145K/D153E/A159V/T366K), detected using chemical cross-linking agents (N-succinimidyl 3-(2-pyridyldithio)propionate and sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate. E. coli BW25113 ΔacrAB ΔtolC210::Tn10 cultures that co-expressed hexa-His-tagged wild-type TolC (WT) or TolC (N145K/D153E/A159V/T366K) and wild-type AcrA (WT) or one of the AcrA cysteine variants (AcrA (D111C) or AcrA (L132C)) are shown. All cultures were treated with N-succinimidyl 3-(2-pyridyldithio)propionate (S), sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (L), or none (−). Affinity-purified TolC and cross-linked AcrA proteins were separated by SDS-PAGE and immunoblotted as indicated. B, physical interaction between the TolC tip and the membrane fusion protein tip regions, revealed by the size-exclusion chromatography with the SDS-PAGE analysis. Panel a, MacA-TolCα hybrid dimer. The result indicates that this protein forms the funnel-like structure because the elution volume is identical to the hexameric Aa MacA (see Fig. 1B). Panel b, wild-type E. coli MacA-AcrAα hybrid. E. coli MacA-AcrAα hybrid, which has AcrA α-hairpin, is eluted as a monomer. Panel c, mixture of E. coli MacA-AcrAα hybrid and MacA-TolCα hybrid dimer. The complex peak (Ap-Tp) and the E. coli MacA-AcrAα hybrid peak (Ap) are indicated in the chromatogram. Ap and Tp stand for MacA-AcrAα hybrid and MacA-AcrAα hybrid dimer, respectively, and Ap-Tp indicates complex between them. See supplemental Fig. S8 for the interaction between E. coli MacA-AcrAα hybrid mutants and MacA-TolCα hybrid dimer. C, representative electron microscopic image of a complex consisting of E. coli MacA-AcrAα hybrid and Aa MacA-TolCα hybrid dimer, which was purified using the size-exclusion chromatography as in Fig. 3B, panel b. The sample was preserved in negative stain (uranyl formate) and imaged at ×80,000. A reference-free class formed from an average of the side views is shown as an inset and can be compared with a projection of the reconstructed density map. D, surface representation of the reconstructed density map displayed in top view, tilted, and side view (top row). The complex model of E. coli MacA-AcrAα hybrid (magenta) and Aa MacA-TolCα hybrid dimer (cyan for the TolC α-barrel tip, and red for the Aa MacA part) is docked into the density map (bottom row).
FIGURE 4.
FIGURE 4.
A TolC and AcrA docking model based on the tip-to-tip interaction. A, ribbon representation (left) and the surface representation (right) of a putative model of the TolC (cyan) and AcrA (blue) complex. The open conformation of TolC was generated by adopting the AcrA α-barrel conformation to the TolC α-barrel end region. It was brought to the top of E. coli AcrA hexamer to establish the intermeshing cogwheel interactions. B, bottom view of TolC in the docked complex displayed in A. The semi-transparent surface representation is shown with the ribbon representation. The central channel is wide open. C, top view of the AcrA hexamer in the docked complex. Structural resemblance to the TolC open structure is shown.
FIGURE 5.
FIGURE 5.
Structural complementarities between the AcrA hexamer model and the substrate exit domain of AcrB trimer and molecular docking of AcrA and TolC. A, surface representations of the AcrA hexamer model. Top, bottom view of the AcrA hexamer. From the distance, the approximate size of the triangular hollow can be deduced. Bottom, side view of the AcrA hexamer. The internal hollow at the funnel stem and the funnel mouth are indicated by red dotted lines. B, surface representations of the AcrB homotrimer using PDB code 2W1B (21). Substrate exit domain is in orange; porter domain is in magenta, and transmembrane (TM) domain is in gray. Top, top view of the AcrB trimer with approximate dimensions of the triangular “substrate exit domain.” The longest length of the porter domain is indicated by 70 Å, which matches the inter-MP domain distance shown in Fig. 1D. Bottom, side view of AcrB trimer. The height of substrate exit domain is indicated. The substrate entrance site is indicated by a yellow arrow and the substrate exit site by a red arrow. C, docking model for AcrA-AcrB complex is displayed by surface representations. The AcrA hexamer model is shown in blue, and the AcrB is colored as in B. Substrate entrance site and the putative substrate exit sites of the complex are indicated by yellow and red arrows, respectively. Top view is shown on the left panel, and the side view is shown on the right. D, docking model for MexA-MexB complex (PDB code 2V50 was used for the MexB structure (35)). Because the MP domain is built in the MexA hexamer model, the docked complex includes the MP domains, which contact the porter domain but do not seem to preclude substrate entrance. Lipid modifications at the N terminus of MexA, which is responsible for the membrane anchorage, are shown.
FIGURE 6.
FIGURE 6.
Assembly model for tripartite efflux pumps. A, interaction of TolC α-barrel tip, AcrA, and AcrB. MacA-TolCα hybrid dimer-coupled resin was prepared using CNBr-activated resin (50 μg; GE Healthcare) and MacA-TolCα hybrid dimer protein (1 mg) according to the manufacturer's instruction. AcrA (residues 26–397; 2 mg/ml) and/or the full-length AcrB with C-terminally hexahistidine tag (His-AcrB; 2 mg/ml) were incubated with the coupled resin (20 μl) for 2 h at 4 °C, and then the resin was thoroughly washed with PBS and applied to SDS-PAGE, followed by Western blotting. To detect AcrA and AcrB, anti-AcrA and anti-His antibodies were used, respectively. B, left, the ribbon representation of the TolC3-AcrA6-AcrB3 complex is colored by its components. The TolC trimer (cyan) contacts the funnel stem of the AcrA hexamer (blue), and the AcrB trimer (orange) contacts the funnel mouth of the AcrA hexamer. In particular, the flexible AcrA MP domains (blue ovoid) make a pair and interact with the porter domain of AcrB, accommodating the dynamic structural movement coupled with the proton translocation and the substrate transport. The substrate moving passage is indicated by a red arrow. The complex spans the entire periplasmic space, inner membrane, and outer membrane. The periplasmic part of the ternary complex is 230 Å long. Right, the ribbon representation of the OprM3-MexA6-MexB3 complex. The OprM trimer (cyan), MexA hexamer (violet), and MexB trimer (orange) make a long complex in the same arrangement with the TolC-AcrA-AcrB complex. The MP domains of MexA are shown. The periplasmic part of the complex is 10 Å shorter because the α-hairpin domain of MexA is shorter than that of AcrA.

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