Antibiotic-sensitive TolC mutants and their suppressors

The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic alpha-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex(+) phenotype. These novel phenotypes of TolC(R367H) and TolC(R390C) were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC(R367H) mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC(R367H), mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.     

A model of a transmembrane drug-efflux pump from Gram-negative bacteria

In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric inner-membrane proton-antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.     

Interaction between the TolC and AcrA proteins of a multidrug efflux system of Escherichia coli

This paper provides the biochemical evidence for physical interactions between the outer membrane component, TolC, and the membrane fusion protein component, AcrA, of the major antibiotic efflux pump of Escherichia coli. Cross-linking between TolC and AcrA was independent of the presence of any externally added substrate of the efflux pump or of the pump protein, AcrB. The biochemical demonstration of a TolC-AcrA interaction is consistent with genetic studies in which extragenic suppressors of a mutant TolC strain were found in the acrA gene.     

Genetic assessment of the role of AcrB β‐hairpins in the assembly of the TolC–AcrAB multidrug efflux pump of Escherichia coli

The tripartite AcrAB–TolC multidrug efflux pump of Escherichia coli is the central conduit for cell‐toxic compounds and contributes to antibiotic resistance. While high‐resolution structures of all three proteins have been solved, much remains to be learned as to how the individual components come together to form a functional complex. In this study, we investigated the importance of the AcrB β‐hairpins belonging to the DN and DC subdomains, which are presumed to dock with TolC, in complex stability and activity of the complete pump. Our data show that the DN subdomain β‐hairpin residues play a more critical role in complex stability and activity than the DC subdomain hairpin residues. The failure of the AcrB DN β‐hairpin deletion mutant to engage with TolC leads to the drug hypersensitivity phenotype, which is reversed by compensatory alterations in the lipoyl and β‐barrel domains of AcrA. Moreover, AcrA and TolC mutants that induce TolC opening also reverse the drug hypersensitivity phenotype of the AcrB β‐hairpin mutants, indicating a failure by the AcrB mutant to interact and thus induce TolC opening on its own. Together, these data suggest that both AcrB β‐hairpins and AcrA act to stabilize the tripartite complex and induce TolC opening for drug expulsion.     

Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps

Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.     

Conformational flexibility in the multidrug efflux system protein AcrA

Intrinsic resistance to multiple drugs in many gram-negative bacterial pathogens is conferred by resistance nodulation cell division efflux pumps, which are composed of three essential components as typified by the extensively characterized Escherichia coli AcrA-AcrB-TolC system. The inner membrane drug:proton antiporter AcrB and the outer membrane channel TolC export chemically diverse compounds out of the bacterial cell, and require the activity of the third component, the periplasmic protein AcrA. The crystal structures of AcrB and TolC have previously been determined, and we complete the molecular picture of the efflux system by presenting the structure of a stable fragment of AcrA. The AcrA fragment resembles the elongated sickle shape of its homolog Pseudomonas aeruginosa MexA, being composed of three domains: beta-barrel, lipoyl, and alpha-helical hairpin. Notably, unsuspected conformational flexibility in the alpha-helical hairpin domain of AcrA is observed, which has potential mechanistic significance in coupling between AcrA conformations and TolC channel opening.     

Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system

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.     

Cross-linked complex between oligomeric periplasmic lipoprotein AcrA and the inner-membrane-associated multidrug efflux pump AcrB from Escherichia coli

In Escherichia coli, the intrinsic levels of resistance to multiple antimicrobial agents are produced through expression of the three-component multidrug efflux system AcrAB-TolC. AcrB is a proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the periplasm and the outer membrane, respectively. In this study, these three proteins were expressed separately, and the interactions between them were analyzed by chemical cross-linking in intact cells. We show that AcrA protein forms oligomers, most probably trimers. In this oligomeric form, AcrA interacts specifically with AcrB transporter independently of substrate and TolC.     

Flexibility in a drug transport accessory protein: molecular dynamics simulations of MexA

Drug resistance in gram-negative bacteria may be conferred via efflux through a tripartite complex of an inner membrane pump, an outer membrane pore, and a periplasmic adaptor protein. These are AcrB, TolC, and AcrA, respectively, in Escherichia coli. In Pseudomonas aerugonisa, their homologs are MexB, OprM, and MexA. Defining the interdomain dynamics of the adaptor protein is essential to understanding the mechanism of complex formation. Extended (25 ns) molecular dynamics simulations of MexA have been performed to determine such interdomain dynamics. Analysis of conformational drift demonstrates substantial motions of the three domains of MexA relative to one another. Principal components analysis reveals a hinge-bending motion and rotation of the alpha-helical hairpin relative to the other domains to be the two dominant motions. These two motions provide an element of considerable flexibility which is likely to be exploited in the adaptor function of MexA.     

Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli.

AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [(14)C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.     

Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode

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.     

Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae

In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB(HI)). Similar to other RND transporters AcrB(HI) associates with AcrA(HI), the periplasmic membrane fusion protein, and the outer membrane channel TolC(HI). Here, we report that AcrAB(HI) confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC(EC)) protein. To investigate the intracellular dynamics of AcrAB(HI), single cysteine mutations were constructed in AcrB(HI) in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB(HI) and in the presence or absence of AcrA(HI) and TolC(EC). We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB(HI).     

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.     

The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC

Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.AcrA, the multidrug efflux protein from Escherichia coli, is the best-characterized member of the membrane fusion protein (MFP) family (24). Periplasmic AcrA associates with the inner-membrane transporter AcrB, belonging to the RND superfamily of proteins, and the outer-membrane factor TolC (22, 23). Together, the three components form a transenvelope multidrug efflux pump responsible for the high levels of intrinsic as well as acquired antibiotic resistance of E. coli.AcrA is anchored into the inner membrane by N-terminal lipid modification. However, genetic complementation studies showed that the presence of the lipid moiety is not required for AcrA function (14, 24). Structural studies of the proteolytically stable core of AcrA (amino acid [aa] residues 46 to 312) and of whole-length MexA, a homologous protein from Pseudomonas aeruginosa, showed that these proteins have modular structures (Fig. ​(Fig.1A).1A). They comprise the α-helical hairpin, the lipoyl-binding domain, and the α-β-barrel domain (2, 9, 14). Mutagenesis and chemical cross-linking studies identified the α-helical hairpin of AcrA as a TolC-binding domain, whereas the α-β-barrel domain was proposed to bind AcrB (6, 11, 12, 16). Surprisingly, in isothermal calorimetry experiments, the core fragment of AcrA without its C-terminal domain (C-domain) was able to bind neither AcrB nor TolC (23). In contrast, the whole-length AcrA interacted with both components. This result suggested that the C-domain of AcrA might be important for these interactions. In crystal structures, however, the C-domains of AcrA and MexA were not resolved, and their structures remain unknown.Open in a separate windowFIG. 1.Proteolytic profiles of AcrA^his in vitro and in vivo. (A) Schematic representation of the secondary structure of AcrA. The unique N-terminal Cys25, which is lipid modified after processing in the periplasm, is shown with an arrow. Positions of amino acid residues that form the α-β-barrel, lipoyl-binding, and α-helical hairpin domains are indicated. AcrA residues cleaved by trypsin are indicated by arrowheads. The 28.9-kDa (K46-R315) core and the 26.5-kDa fragment (K46-R294) are also indicated. (B) Purified AcrA^his (final concentration, 1.95 μM) was digested with trypsin (final concentration, 0.10 μM) at 37°C. Aliquots (10 μl) were taken at different time points, and reactions were terminated by boiling in the SDS sample buffer for 5 min. Tryptic fragments were resolved by SDS-PAGE and analyzed by silver nitrate staining. Minor fragments in the untreated control (0 min) are contaminants that copurify with AcrA^his. Lane M, molecular marker. (C) Proteolytic profiles of AcrA^his in E. coli AG100AX cells carrying pA^his and pA^hisB plasmids. After treatment with increasing concentrations of trypsin for 60 min at 37°C, the whole-cell proteins were resolved by SDS-PAGE and analyzed by immunoblotting with a polyclonal anti-AcrA antibody. Masses of tryptic fragments of the C-domain of AcrA^his identified by mass spectrometry and by mobility in SDS-PAGE are indicated. O.D., optical density as determined by absorbance at 600 nm.The alignment of sequences of highly diverse MFPs from both gram-negative and gram-positive bacteria showed that amino acid sequences of the C-domains are conserved among members of the MFP family (4). In addition, several studies suggested that this region is important for the function of AcrA. The deletion mutant of AcrA lacking 85 C-terminal aa residues is poorly expressed and nonfunctional in multidrug efflux (14). The replacement of aa 290 to 357 of AcrA with an analogous region of YhiU disrupted AcrA function possibly because of the loss of interaction with the AcrB transporter (5). Random mutagenesis of MexA identified C-terminal amino acid residues as important for MexA oligomerization and interaction with MexB (16, 17).In this study, we identified proteolytically labile sites in the C-domain (aa 315 to 397) of the purified AcrA and compared the accessibility of these sites to that in free AcrA and when engaged in the bipartite and tripartite AcrA, AcrB, and TolC interactions in vivo. We found that the assembly of the AcrAB-TolC complex, but not bipartite AcrA-AcrB and AcrA-TolC interactions, protects the C-domain of AcrA from proteolytic digestion. This result suggested that this domain of AcrA interacts with AcrB, TolC, or both. The functional significance of the C-domain was confirmed by site-directed mutagenesis. A single G363C substitution significantly impairs the multidrug efflux activity of AcrAB-TolC.     

Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump

AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that, among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues 357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at, its C terminus is involved in the interaction with its cognate pump protein, AcrB.     

Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops

AcrAB-TolC is a constitutively expressed, tripartite efflux transporter complex that functions as the primary resistance mechanism to lipophilic drugs, dyes, detergents, and bile acids in Escherichia coli. TolC is an outer membrane channel, and AcrA is an elongated lipoprotein that is hypothesized to span the periplasm and coordinate efflux of such substrates by AcrB and TolC. AcrD is an efflux transporter of E. coli that provides resistance to aminoglycosides as well as to a limited range of amphiphilic agents, such as bile acids, novobiocin, and fusidic acid. AcrB and AcrD belong to the resistance nodulation division superfamily and share a similar topology, which includes a pair of large periplasmic loops containing more than 300 amino acid residues each. We used this knowledge to test several plasmid-encoded chimeric constructs of acrD and acrB for substrate specificity in a marR1 DeltaacrB DeltaacrD host. AcrD chimeras were constructed in which the large, periplasmic loops between transmembrane domains 1 and 2 and 7 and 8 were replaced with the corresponding loops of AcrB. Such constructs provided resistance to AcrB substrates at levels similar to native AcrB. Conversely, AcrB chimeras containing both loops of AcrD conferred resistance only to the typical substrates of AcrD. These results cannot be explained by simply assuming that AcrD, not hitherto known to interact with AcrA, acquired this ability by the introduction of the loop regions of AcrB, because (i) both AcrD and AcrA were found, in this study, to be required for the efflux of amphiphilic substrates, and (ii) chemical cross-linking in intact cells efficiently produced complexes between AcrD and AcrA. Since AcrD can already interact with AcrA, the alterations in substrate range accompanying the exchange of loop regions can only mean that substrate recognition (and presumably binding) is determined largely by the two periplasmic loops.     

Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist

Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.     

Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking

The AcrAB-TolC system exports a wide variety of drugs and toxic compounds, and confers intrinsic drug tolerance on Escherichia coli. The crystal structures suggested that AcrB and TolC directly dock with each other. However, biochemical and biophysical evidence of their interaction has been contradictory until recently. In this study, we examine the interaction sites by means of in vivo disulfide cross-linking between cysteine residues introduced by site-directed mutagenesis at the tops of the vertical hairpins of AcrB and the bottoms of the coiled coils of polyhistidine-tagged TolC molecules, which are structurally predicted docking sites. The AcrB-TolC complex formed through disulfide cross-linking was detected when a specific pair of mutants was coexpressed in E. coli. Our observations suggested that the AcrB-TolC complex may be formed through a two-step mechanism via transient tip-to-tip interaction of AcrB and TolC. The cross-linking was not affected by AcrA, the substrate, or a putative proton coupling site mutation.     

AcrAB and related multidrug efflux pumps of Escherichia coli

The AcrAB system of Escherichia coli is a multidrug efflux system composed of an RND-type transporter AcrB and a periplasmic accessory protein AcrA, and pumps out a wide variety of lipophilic and amphiphilic inhibitors directly into the medium, presumably through the TolC outer membrane channel. AcrA, a highly elongated protein, is thought to bring the outer and inner membranes closer. It forms a trimer that interacts with a monomeric AcrB, which was shown by in vitro reconstitution to be a proton antiporter. Details of interaction between the     

Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.