Structure of a mutated TolC
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Abstract Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae , binds TolC of a related species ( K. quasipneumoniae ) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.
Efflux
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Inner membrane
Colicin
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Summary The major Escherichia coli multidrug efflux pump AcrAB–TolC expels a wide range of antibacterial agents. Using in vivo cross‐linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate‐induced assembly of the closely related TolC‐dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross‐linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo . Confirming this view, AcrA could be cross‐linked independently to AcrB and TolC in vivo , and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C‐terminal half of AcrA, but binding to TolC required interaction of N‐ and C‐terminal polypeptides spanning the lipoyl‐like domains predicted to present the intervening coiled‐coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug‐independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.
Translocase
Efflux
Inner membrane
Transport protein
Multidrug Resistance-Associated Proteins
Isothermal Titration Calorimetry
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ABSTRACT The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB -deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCE The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB -deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.
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Inner membrane
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TolC and the other members of the outer membrane factor (OMF) family are outer membrane proteins forming trimeric channels that serve as a conduit for most actively effluxed substrates in Gram-negative bacteria by providing a key component in a multitude of tripartite efflux-pumps. Current models of tripartite pump assembly ascribe substrate selection to the inner-membrane transporter and periplasmic-adapter protein (PAP) assembly, suggesting that TolC is a passive, non-selective channel. While the membrane-embedded portion of the protein adopts a porin-like fold, the periplasmic domain of TolC presents a unique "alpha-barrel" architecture. This alpha-barrel consists of pseudo-continuous α-helices forming curved coiled-coils, whose tips form α-helical hairpins, relaxation of which results in a transition of TolC from a closed to an open-aperture state allowing effective efflux of substrates through its channel. Here, we analysed the effects of site-directed mutations targeting the alpha-barrel of TolC, of the principal tripartite efflux-pump Escherichia coli AcrAB-TolC, on the activity and specificity of efflux. Live-cell functional assays with these TolC mutants revealed that positions both at the periplasmic tip of, and partway up the TolC coiled-coil alpha-barrel domain are involved in determining the functionality of the complex. We report that mutations affecting the electrostatic properties of the channel, particularly the D371V mutation, significantly impact growth even in the absence of antibiotics, causing hyper-susceptibility to all tested efflux-substrates. These results suggest that inhibition of TolC functionality is less well-tolerated than deletion of tolC, and such inhibition may have an antibacterial effect. Significantly and unexpectedly, we identified antibiotic-specific phenotypes associated with novel TolC mutations, suggesting that substrate specificity may not be determined solely by the transporter protein or the PAP, but may reside at least partially with the TolC-channel. Furthermore, some of the effects of mutations are difficult to reconcile with the currently prevalent tip-to-tip model of PAP-TolC interaction due to their location higher-up on the TolC alpha-barrel relative to the proposed PAP-docking sites. Taken together our results suggest a possible new role for TolC in vetting of efflux substrates, alongside its established role in tripartite complex assembly.
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Efflux
Transport protein
Gram-Negative Bacteria
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VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.
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Inner membrane
Multidrug Resistance-Associated Proteins
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ABSTRACT The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli , was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914–919, 2000), demonstrating a distinctive architecture comprising outer membrane β-barrel and periplasmic helical-barrel structures, which assemble differently from the common β-barrel-only conformation of porins. Based on their sequence similarity, a similar content of α-helical and β-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the “iris” mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.
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Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel. Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.
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Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.
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Inner membrane
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