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, 287 (15), 11740-50

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

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Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

Yongbin Xu et al. J Biol Chem.

Abstract

Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

Figures

FIGURE 1.
FIGURE 1.
Structure of A. actinomycetemcomitans MacA-MexAα hybrid. A, schematic diagram of construction of A. actinomycetemcomitans MacA-MexAα hybrid. The α-hairpin domain of A. actinomycetemcomitans MacA (residues 89–180) is substituted with that of P. aeruginosa MexA (residues 95–158). B, crystal packing of A. actinomycetemcomitans MacA-MexAα hybrid, compared with that of A. actinomycetemcomitans MacA. Left, the asymmetric unit of A. actinomycetemcomitans MacA-MexAα hybrid is composed of a pair of dimeric units of the protein, which were colored yellow, red, green, or blue. The crystallographic 3-fold symmetry-related molecules are shown in gray. Right, the asymmetric unit of A. actinomycetemcomitans MacA is colored red, and the molecules generated by the crystallographic 6- and 2-fold are shown in gray. In both crystals, the hexameric units pack with tip-to-tip interactions. C, the hexameric structure of A. actinomycetemcomitans MacA-MexAα hybrid. This chimeric protein consists of an α-hairpin domain from P. aeruginosa MexA (green) and lipoyl, β-barrel, and membrane proximal (MP) domains, shown in gray, from A. actinomycetemcomitans MacA. The side view is displayed in the left panel, and the top view is shown in the right panel.
FIGURE 2.
FIGURE 2.
Construction and structural models of A. actinomycetemcomitans MacA-OprMα hybrid dimer. A, schematic diagram of construction of A. actinomycetemcomitans MacA-OprMα hybrid dimer. Two units of A. actinomycetemcomitans MacA are linked, and each A. actinomycetemcomitans MacA unit contains either the OprM α-hairpin tip region in repeat 1 (residues 189–212) or the tip region in repeat 2 (residues 396–419) instead of the corresponding region of A. actinomycetemcomitans MacA α-hairpin tip (residues 124–147). The amino acid sequence and the heptad positions are shown in the bottom panel. The red box indicates the conserved (V/T)GV motifs. B, molecular modeling of the chimeric protein A. actinomycetemcomitans MacA-OprMα hybrid dimer. The amino acids derived from OprM α-barrel tip regions are colored blue for repeat 1 and purplish blue for repeat 2. C, Top views of the chimeric protein of the A. actinomycetemcomitans MacA-OprMα hybrid dimer (left) and the crystal structure of OprM in the closed state (right). The channel of the chimeric protein is wide open at the OprM tip region. The residues involved in channel closing are shown in the stick representations. MP, membrane proximal.
FIGURE 3.
FIGURE 3.
Binding between MexA α-hairpin and OprM α-barrel tip regions. A, the physical interaction between the OprM α-barrel tip and the MexA α-barrel tip regions as revealed by size exclusion chromatography using SDS-PAGE analysis. i, A. actinomycetemcomitans MacA-OprMα hybrid dimer protein (Op) alone. The result indicates that this protein forms a funnel-like structure as the elution volume is identical to hexameric A. actinomycetemcomitans MacA (data not shown). ii, A. actinomycetemcomitans MacA-MexAα hybrid protein (Mp) alone. The profile indicates that the protein is eluted as a monomer. iii, mixture of A. actinomycetemcomitans MacA-OprMα hybrid dimer and A. actinomycetemcomitans MacA-MexAα hybrid proteins. The peaks in the chromatogram indicate the complex (Cp), A. actinomycetemcomitans MacA-MexAα hybrid (Mp), and MacA-OprMα hybrid dimer (Op). According to the standard calibration curve for the size exclusion chromatographic column, MacA-OprMα hybrid dimer, A. actinomycetemcomitans MacA-MexAα hybrid, and the complex was calculated as 120, 61, and 670 kDa, respectively. B, the interaction of P. aeruginosa OprM α-barrel tip or E. coli TolC α-barrel tip with the α-hairpin domains of MexA or AcrA. MacA-AcrAα hybrid that contains AcrA α-hairpin domain (Ec AcrAα) (14) or MacA-MexAα hybrid (Pa MexAα) was covalently linked to agarose using CNBr-activated resin (50 μg; GE Healthcare) according to the manufacturer's instructions. Then, MacA-TolCα hybrid dimer protein (Ec TolCα tip; 1 mg) or MacA-OprMα hybrid dimer protein (Pa OprMα tip; 1 mg) was incubated with the agarose resin for 2 h at 4 °C, followed by thorough washing with PBS, and then application by SDS-PAGE. The asterisk indicates that the proteins were liberated partially from the resin by denaturation, even though the proteins were covalently attached to the resin, due to the multimeric structure of the proteins. Because a part of the MacA-OprMα hybrid dimer protein did not form the hexameric structure (i), the amount of the bound protein is relatively small compared with the MacA-TolCα hybrid dimer protein. However, the bindings of the protein to AcrA α-barrel and MexA α-barrel are strong and specific because the MacA-OprMα hybrid dimer protein is not bound to the resin in the absence of the bait proteins.
FIGURE 4.
FIGURE 4.
Electron microscopic analysis of the binding interface between MexA and OprM. A, representative single particles of A. actinomycetemcomitans MacA-MexAα hybrid and A. actinomycetemcomitans MacA-OprMα hybrid dimer, preserved in negative stain (uranyl-formate) and imaged at 80,000× magnification. Reference-free class averages reveal flexibility in one of the two ends (arrows), whereas the other end is stable. B, surface representations of the reconstructed density map from electron microscope images, displayed in a side (i), top (ii), bottom (iii), horizontal section (iv), and vertical section views (v). The central barrel and its bulge are indicated by a line and arrows. The 10-Å scale bar is shown, indicating that the length of the bulge is ∼3 Å. 2d, two-dimensional.
FIGURE 5.
FIGURE 5.
Modeled structures for MexA α-hairpin and OprM α-barrel tip regions, docked to the electron density map. A, the hexameric structure of A. actinomycetemcomitans MacA-MexAα hybrid (shown in green) and the hexameric model of A. actinomycetemcomitans MacA-OprMα hybrid dimer (shown in violet) were fitted to the electron density map. The MexA α-hairpin and OprM α-barrel tip regions are indicated. The arrow indicates the bulged region in the electron density map whose shape matches well with the location of the binding interface between MexA α-hairpin and OprM α-barrel tip regions. B, stick representations at the interface between MexA α-hairpin (shown in green) and OprM α-barrel tip region (shown in blue for repeat 1 and violet for repeat 2). The conserved amino acid residues, Arg-119, Leu-123, and Ser-130 of MexA are shown as cyan. The bottom panel shows a sequence alignment on the α-hairpin tip region of P. aeruginosa (Pa) MexA, E. coli (Ec) AcrA, E. coli MacA, and A. actinomycetemcomitans MacA where the key conserved amino acid residues Arg, Leu, and Ser are highlighted. C, close-up view of MexA Arg-119-mediated interactions. The van der Waals radius of MexA Arg-119 is displayed using dots. The red dotted line indicates hydrogen bonding between the guanidine group of MexA Arg-119 and the OprM backbone carbonyl group. The OprM tip regions are shown as violet or blue and covered by a semitransparent surface representation. The MexA residues are colored green or dark green. The long aliphatic chain of Arg-119 occupies the space between the two proteins, and the terminal group makes a polar interaction. D, a close-up view of MexA Ser-130-mediated interaction. MexA Ser-130 makes a hydrogen bond with OprM Ser-138 in repeat 1 and with OprM Asn-410 in repeat 2 (not shown). E, a close-up view of MexA Leu-123-mediated interactions. The conserved Leu-123 and Leu-122 residues make a leucine-zipper-like interaction with the OprM (V/T)GV motif in both repeats 1 and 2.
FIGURE 6.
FIGURE 6.
The synergistic binding of AcrA and TolC to E. coli peptidoglycan. The full-length AcrA, except for the signal peptide and lipid modification (AcrA; 80 μg) and/or the full-length TolC with the C-terminally hexahistidine tag (TolC-His; 70 μg) were incubated with the purified peptidoglycans (PGN; 200 μg) from E. coli or S. aureus in an insoluble form. After washing with PBS, the peptidoglycans were applied to SDS-PAGE and analyzed by Western blot with the indicating antibodies.

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