The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity (P(K)/P(Cl)= approximately 3), whereas TolC is more selective to cations (P(K)/P(Cl)= approximately 30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.     

The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity (P_K/P_Cl = ∼ 3), whereas TolC is more selective to cations (P_K/P_Cl = ∼ 30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.     

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.     

Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals

TolC is an outer membrane protein required for the export of virulence proteins and toxic compounds without a periplasmic intermediate. We show that TolC is an integral part of the translocator, interacting with inner membrane components, by demonstrating a need for TolC in protein export not only from intact cells but also from sphaeroplasts. To establish the structure of TolC, and thus gain information on how this might be achieved, the protein was purified from the Escherichia coli outer membrane, as a trimer, and crystallized in two-dimensional lattices by reconstitution in phospholipid bilayers. The projection structure at 12 Å resolution showed a threefold symmetric molecule of 58 Å outer diameter, and a single pool of stain filling its centre. Side views parallel to the membrane plane revealed an additional domain outside the membrane. Eighteen membrane-spanning β-strands were predicted for the 51.5 kDa monomer, excluding a 7 kDa C-terminal segment, and this segment was shown to contain a proteinase K-sensitive site that was exposed in reconstituted membranes and sphaeroplasts, but which was protected in intact cells. The combined data suggest that TolC is a trimeric outer membrane protein with each monomer comprising a membrane domain, predicted to be β-barrel, and a C-terminal periplasmic domain. The latter could form part of the bridge to the energized inner membrane component of the translocation complex.     

Multiple conformational states and gate opening of outer membrane protein TolC revealed by molecular dynamics simulations

Outer membrane protein TolC serves as an exit duct for exporting substances out of cell. The occluded periplasmic entrance of TolC is required to open for substrate transport, although the opening mechanism remains elusive. In this study, systematic molecular dynamics (MD) simulations for wild type TolC and six mutants were performed to explore the conformational dynamics of TolC. The periplasmic gate was shown to sample multiple conformational states with various degrees of gating opening. The gate opening was facilitated by all mutations except Y362F, which adopts an even more closed state than wild type TolC. The interprotomer salt‐bridge R367–D153 is turned out to be crucial for periplasmic gate opening. The mutations that disrupt the interactions at the periplasmic tip may affect the stability of the trimeric assembly of TolC. Structural asymmetry of the periplasmic gate was observed to be opening size dependent. Asymmetric conformations are found in moderately opening states, while the most and the least opening states are often more symmetric. Finally, it is shown that lowering pH can remarkably stabilize the closed state of the periplasmic gate. Proteins 2014; 82:2169–2179. © 2014 Wiley Periodicals, Inc.     

Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC

The outer membrane channel TolC is a key component of multidrug efflux and type I secretion transporters in Escherichia coli. Mutational inactivation of TolC renders cells highly susceptible to antibiotics and leads to defects in secretion of protein toxins. Despite impairment of various transport functions, no growth defects were reported in cells lacking TolC. Unexpectedly, we found that the loss of TolC notably impairs cell division and growth in minimal glucose medium. The TolC‐dependent phenotype was further exacerbated by the loss of ygiB and ygiC genes expressed in the same operon as tolC and their homologues yjfM and yjfC located elsewhere on the chromosome. Our results show that this growth deficiency is caused by depletion of the critical metabolite NAD⁺ and high NADH/NAD⁺ ratios. The increased amounts of PspA and decreased rates of NADH oxidation in ΔtolC membranes indicated stress on the membrane and dissipation of a proton motive force. We conclude that inactivation of TolC triggers metabolic shutdown in E. coli cells grown in minimal glucose medium. The ΔtolC phenotype is partially rescued by YgiBC and YjfMC, which have parallel functions independent from TolC.     

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.     

Surface charges on the outer side of mollusc neuron membrane

Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem. 30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK _Ca=90±10,K _Sr=60±10,K _Ba=25±5 andK _Mg=16±5m ^–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK _Ca=67±10,K _Sr=20±5 andK _Ba=19±5m ^–1 at pH=7.0. The same groups bind H⁺ ions with apparent pK=6.2±0.2 that corresponds toK _H=1.6×10⁶ m ^–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm² (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm² (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents.     

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

On the role of TolC in multidrug efflux: the function and assembly of AcrAB–TolC tolerate significant depletion of intracellular TolC protein

TolC channel provides a route for the expelled drugs and toxins to cross the outer membrane of Escherichia coli. The puzzling feature of TolC structure is that the periplasmic entrance of the channel is closed by dense packing of 12 α‐helices. Efflux pumps exemplified by AcrAB are proposed to drive the opening of TolC channel. How interactions with AcrAB promote the close‐to‐open transition in TolC remains unclear. In this study, we investigated in vivo the functional and physical interactions of AcrAB with the closed TolC and its conformer opened by mutations in the periplasmic entrance. We found that the two conformers of TolC are readily distinguishable in vivo by characteristic drug susceptibility, thiol modification and proteolytic profiles. However, these profiles of TolC variants respond neither to the in vivo stoichiometry of AcrAB:TolC nor to the presence of vancomycin, which is used often to assess the permeability of TolC channel. We further found that the activity and assembly of AcrAB–TolC tolerates significant changes in amounts of TolC and that only a small fraction of intracellular TolC is likely used to support efflux needs of E. coli. Our findings explain why TolC is not a good target for inhibition of multidrug efflux.     

Generation of daunomycin radicals on the outer side of the erythrocyte membrane

The generation of the daunomycin semiquinone was studied in intact red blood cells under CO atmosphere by ESR spectroscopy. The undialyzed hemolysates and the spin broadening agent chromium oxalate quenched the ESR signal, suggesting external location of the ESR-detectable radicals and their slow diffusion inside. A constant outward flow of O2- was detected by monitoring the approach to the steady state of the ESR signal of Cu,Zn superoxide dismutase externally added to red blood cells plus daunomycin in air. This suggests a reductase on the outer side of the erythrocyte membrane as the source of daunomycin radicals.     

Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase

Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils. These changes converge to the native MexAB partner OprM, and indicate an interface key to the function and diversity of efflux pumps.     

Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue

The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP-binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli , TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi . By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed-milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti-TolC antibodies cross-reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens . hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug- and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.     

Type I secretion and multidrug efflux: transport through the TolC channel-tunnel

The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm. The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules. The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known.     

Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25

A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.     

OmpT: molecular dynamics simulations of an outer membrane enzyme

Five molecular dynamics simulations (total duration >25 ns) have been performed on the Escherichia coli outer membrane protease OmpT embedded in a dimyristoylphosphatidylcholine lipid bilayer. Globally the protein is conformationally stable. Some degree of tilt of the beta-barrel is observed relative to the bilayer plane. The greatest degree of conformational flexibility is seen in the extracellular loops. A complex network of fluctuating H-bonds is formed between the active site residues, such that the Asp210-His212 interaction is maintained throughout, whereas His212 and Asp83 are often bridged by a water molecule. This supports a catalytic mechanism whereby Asp83 and His212 bind a water molecule that attacks the peptide carbonyl. A configuration yielded by docking calculations of OmpT simulation snapshots and a model substrate peptide Ala-Arg-Arg-Ala was used as the starting point for an extended Huckel calculation on the docked peptide. These placed the lowest unoccupied molecular orbital mainly on the carbon atom of the central C=O in the scissile peptide bond, thus favoring attack on the central peptide by the water held by residues Asp83 and His212. The trajectories of water molecules reveal exchange of waters between the intracellular face of the membrane and the interior of the barrel but no exchange at the extracellular mouth. This suggests that the pore-like region in the center of OmpT may enable access of water to the active site from below. The simulations appear to reveal the presence of specific lipid interaction sites on the surface of the OmpT barrel. This reveals the ability of extended MD simulations to provide meaningful information on protein-lipid interactions.     

Assembly of TolC, a structurally unique and multifunctional outer membrane protein of Escherichia coli K-12

TolC is a multifunctional outer membrane protein of Escherichia coli that folds into a novel alpha-beta-barrel conformation absent in the other model outer membrane proteins used in assembly studies. The data presented in this work show that the unique folded structure of TolC reflects a unique assembly pathway. During its assembly, the newly translocated nascent TolC monomers are released in the periplasm. Maturation of these nascent monomers, and possibly their oligomerization, in the periplasm precedes their insertion in the outer membrane. The completion of the assembly process is signaled by the development of a characteristic proteinase K-resistant fragment generated by cleavage at a single, periplasmically exposed, protease-sensitive site of the membrane-anchored trimer. None of the assembly steps of TolC is affected by known folding factors, such as SurA, Skp, and lipopolysaccharide, which have profound effects on the assembly of other model trimeric outer membrane proteins. Two assembly-defective TolC mutants were isolated and characterized. One of the mutants (TolC(I106N)) was defective in the folding of nascent monomers, while the other (TolC(S350F)) was impaired in steps involving trimerization and membrane insertion of folded monomers.     

Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC

The trimeric TolC protein of Escherichia coli comprises an outer membrane beta-barrel and a contiguous alpha-helical barrel projecting across the periplasm. This provides a single 140 A long pore for multidrug efflux and protein export. We have previously reported that trivalent cations such as hexammine cobalt can severely inhibit the conductivity of the TolC pore reconstituted in planar lipid bilayers. Here, isothermal calorimetry shows that Co(NH(3))(6)(3+) binds to TolC with an affinity of 20 nM. The crystal structure of the TolC-Co(NH(3))(6)(3+) complex was determined to 2.75 A resolution, and showed no significant difference in the protein when compared with unliganded TolC. An electron density difference map revealed that a single ligand molecule binds at the centre of the periplasmic entrance, the sole constriction of TolC. The octahedral symmetry of the ligand and the three-fold rotational symmetry of the TolC entrance determine a binding site in which the ligand forms hydrogen bonds with the Asp(374) residue of each monomer. When Asp(374) was substituted by alanine, high affinity ligand binding was abolished and inhibition of TolC pore conductivity in lipid bilayers was alleviated. Comparable effects followed independent substitution of the neighbouring Asp(371), indicating that this aspartate ring also contributes to the high affinity ligand binding site. As the electronegative entrance is widely conserved in the TolC family, it may be a useful target for the development of inhibitors against multidrug resistant pathogenic bacteria.