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.     

Unidirectional peristaltic movement in multisite drug binding pockets of AcrB from molecular dynamics simulations

Tripartite complex AcrB-ToIC, the major efflux system in Escherichia coli, is the principal multidrug transporter in Gram-negative bacteria, which is important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel ToIC and the membrane fusion protein AcrA. Recently, the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin were reported. Here we performed 20 ns molecular dynamics (MD) simulations of the AcrB-rifampicin-minocycline complex in a lipid bilayer and explicit water. We found that the bound drugs, rifampicin and erythromycin, made a unidirectional peristaltic movement towards the extrusion funnel of ToIC, which was facilitated by the water efflux in the channel of AcrB. With a shift of the Phe-617 loop, rifampicin in the access monomer moved towards the entrance of the distal binding pocket. Minocycline in the binding monomer moved from the distal binding pocket towards the gate of the central funnel. The channel between the entrance and the gate made a concerted opening during the MD simulations, which was helpful for the peristaltic movement. Our results showed that the mutations of Gly616Pro and Gly619Pro prevented the movement of the Phe-617 loop, which indicated the critical role of the flexibility of the Phe-617 loop. In addition, three putative proton translocation channels were proposed based on our results. Our study provided dynamical information and important residues for the peristaltic movement in AcrB, which were critical for substrate uptake and extrusion function.     

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.     

Determination of real-time efflux phenotypes in Escherichia coli AcrB binding pocket phenylalanine mutants using a 1,2'-dinaphthylamine efflux assay

To evaluate the importance of phenylalanine residues for substrate transport in the Escherichia coli efflux pump protein AcrB, we subjected Phe-to-Ala binding pocket mutants to a real-time efflux assay with the novel near-infrared lipophilic membrane probe 1,2'-dinaphthylamine (1,2'-DNA). All mutations, with the exception of F617A, led to considerable retardation of efflux. F610A was the point mutation with the most pronounced impact, followed by F628A, F615A, F136A, and F178A. This is the first study to demonstrate the importance of single phenylalanine residues within the AcrB binding pocket for real-time substrate transport.     

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.     

Vestibules are part of the substrate path in the multidrug efflux transporter AcrB of Escherichia coli

The path of substrates in the multidrug efflux pump AcrB of Escherichia coli was examined by using labeling with a lipophilic substrate mimic, Bodipy FL maleimide. Four (out of eight) residues in the vestibule bound the dye, suggesting its role in substrate transport, whereas only one (out of nine) residue in the central cavity tested positive.     

New insights into the structural and functional involvement of the gate loop in AcrB export activity

AcrB is a major multidrug exporter in Escherichia coli and other Gram-negative bacteria. Its gate loop, located between the proximal and the distal pockets, have been reported to play important role in the export of many antibiotics. This loop location, rigidity and interactions with substrates have led recent reports to suggest that AcrB export mechanism operates in a sequential manner. First the substrate binds the proximal pocket in the access monomer, then it moves to bind the distal pocket in the binding monomer and subsequently it is extruded in the extrusion monomer. Recently, we have demonstrated that the gate loop is not required for the binding of Erythromycin but the integrity of this loop is important for an efficient export of this substrate. However, here we show that the antibiotic susceptibilities of the same AcrB gate loop mutants for Doxorubicin were unaffected, suggesting that this loop is not required for its export, and we demonstrate that this substrate may use principally the tunnel-1, located between transmembranes 8 and 9, more often than previously reported. To further explain our findings, here we address the gate loop mutations effects on AcrB solution energetics (fold, stability, molecular dynamics) and on the in vivo efflux of Erythromycin and Doxorubicin. Finally, we discuss the efflux and the discrepancy between the structural and the functional experiments for Erythromycin in these gate loop mutants.     

Multidrug-exporting secondary transporters

The major cause of intrinsic drug resistance in Gram-negative bacteria is a resistance nodulation division type multidrug exporter, which couples with an outer membrane channel and a membrane fusion protein and exports drugs out of the cell, bypassing the periplasm; this process is driven by proton motive force. A recent crystal structure determination of a major resistance nodulation division type multidrug exporter, AcrB in Escherichia coli, greatly advances our understanding of the multidrug export mechanism. The most striking feature of the AcrB trimer is the presence of three vestibules open to the periplasm at the boundary between the periplasmic headpiece and the transmembrane region. Substrates can gain access to the central cavity from the periplasmic surface of the cytoplasmic membrane and are then actively transported through the extramembrane pore into the outer membrane channel TolC, via the funnel at the top of the AcrB headpiece.     

Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution

AcrB is an inner membrane resistance-nodulation-cell division efflux pump and is part of the AcrAB–TolC tripartite efflux system. We have determined the crystal structure of AcrB with bound Linezolid at a resolution of 3.5 Å. The structure shows that Linezolid binds to the A385/F386 loops of the symmetric trimer of AcrB. A conformational change of a loop in the bottom of the periplasmic cleft is also observed.     

Site-directed disulfide cross-linking shows that cleft flexibility in the periplasmic domain is needed for the multidrug efflux pump AcrB of Escherichia coli

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.     

Different functions of MdtB and MdtC subunits in the heterotrimeric efflux transporter MdtB(2)C complex of Escherichia coli

In contrast to homotrimeric transporters of the RND (resistance-nodulation-division) superfamily, which often conduct efflux transport of a wide range of substrates by the functionally rotating mechanism, the mechanism utilized by the heterotrimeric members of this family, which also perform multidrug efflux, is unclear. We examined one heterotrimeric transporter, the MdtB(2)C complex of Escherichia coli, by an extensive cysteine scanning mutagenesis of residues likely involved in ligand transport. Many such mutations in MdtC strongly decreased the level of cloxacillin transport, whereas mutations of corresponding residues in MdtB did not affect transport. Furthermore, many such residues in MdtC were covalently modified by fluorescein maleimide, which acted as a substrate and presumably produced labeling of the residues in the substrate path. In contrast, few residues in MdtB were labeled. Together with the previous data showing that the inactivation of proton translocation channel in MdtC has an only modest effect on transport yet in MdtB totally inactivated the activity, these results suggest that the two subunits, MdtB and MdtC, play very different roles, MdtC likely involved in substrate binding and transport and MdtB presumably inducing the conformational change needed for transport through proton translocation. Three-dimensional models of MdtB and MdtC, based on sequence homology with the AcrB transporter, also support this interpretation.     

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.     

Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS

The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.     

β-Lactam Selectivity of Multidrug Transporters AcrB and AcrD Resides in the Proximal Binding Pocket

β-Lactams are mainstream antibiotics that are indicated for the prophylaxis and treatment of bacterial infections. The AcrA-AcrD-TolC multidrug efflux system confers much stronger resistance on Escherichia coli to clinically relevant anionic β-lactam antibiotics than the homologous AcrA-AcrB-TolC system. Using an extensive combination of chimeric analysis and site-directed mutagenesis, we searched for residues that determine the difference in β-lactam specificity between AcrB and AcrD. We identified three crucial residues at the “proximal” (or access) substrate binding pocket. The simultaneous replacement of these residues in AcrB by those in AcrD (Q569R, I626R, and E673G) transferred the β-lactam specificity of AcrD to AcrB. Our findings indicate for the first time that the difference in β-lactam specificity between AcrB and AcrD relates to interactions of the antibiotic with residues in the proximal binding pocket.     

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.     

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.     

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.     

Identification and characterization of carbapenem binding sites within the RND-transporter AcrB

Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.     

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).