Assembly and channel opening in a bacterial drug efflux machine

Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.     

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 ? into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system. Electronic Publication     

Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB(6)-CusA(3). We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.     

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

Gram-negative bacteria frequently expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membranes. The three parts are the inner membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (MFP, or adaptor); and an outer membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We previously described the crystal structures of both the inner membrane transporter CusA and the MFP CusB of Escherichia coli. We also determined the co-crystal structure of the CusBA adaptor-transporter efflux complex, showing that the transporter CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. Here, we summarize the structural information of these efflux proteins, and present the accumulated evidence that this efflux system uses methionine residues to bind and export Cu(I) and Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and the cytoplasm. We propose a stepwise shuttle mechanism for this pump to export metal ions from the cell.     

Solute channels of the outer membrane: from bacteria to chloroplasts

Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of beta-barrels. Although most of the OEP channels are formed by beta-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP 'portrait gallery'.     

The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 A resolution

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.     

Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.     

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

Multidrug resistance mechanisms: drug efflux across two membranes

A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.     

A constituent of the chloroplast import complex represents a new type of GTP-binding protein

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [α-³²P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.     

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

A crosslinking study reveals novel insights into how the chaperone TAM helps Gram-negative bacteria insert the drug efflux pump subunit TolC into their outer membrane. Bioinformatic analyses show that TolC-like proteins can be found in all LPS-containing bacteria, but also in some monodermic Firmicutes.     

Expression and biochemical characterization of the periplasmic domain of bacterial outer membrane porin TdeA

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic alpha- helical barrel domain and a membrane-embedded beta-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membraneembedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family can fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.     

Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents. A major contribution to this intrinsic multidrug resistance is provided by a number of broadly-specific multidrug efflux systems, including MexAB-OprM and MexXY-OprM. In addition, these and two additional tripartite efflux systems, MexCD-OprJ and MexEF-OprN, promote acquired multidrug resistance as a result of mutational hyperexpression of the efflux genes. In addition to antibiotics, these pumps promote export of numerous dyes, detergents, inhibitors, disinfectants, organic solvents and homoserine lactones involved in quorum sensing. The efflux pump proteins are highly homologous and consist of a cytoplasmic membrane-associated drug-proton antiporter of the Resistance-Nodulation-Division (RND) family, an outer membrane channel-forming protein [sometimes called outer membrane factor (OMF)] and a periplasmic membrane fusion protein (MFP). Homologues of these systems have been described in Stenotrophomonas maltophilia, Burkholderia cepacia, Burkholderia pseudomallei and the non-pathogen Pseudomonas putida, where they play a role in export of and resistance to multiple antimicrobial agents and/or organic solvents. Although the natural function of these multidrug efflux systems is largely unknown, their contribution to antibiotic resistance and their conservation in a number of important human pathogens makes them logical targets for therapeutic intervention.     

Structure–function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD

In Gram-negative bacteria, trans-envelope efflux pumps have periplasmic membrane fusion proteins (MFPs) as essential components. MFPs act as mediators between outer membrane factors (OMFs) and inner membrane factors (IMFs). In this study, structure–function relations of the ATP-driven glycolipid efflux pump DevBCA-TolC/HgdD from the cyanobacterium Anabaena sp. PCC 7120 were analyzed. The binding of the MFP DevB to the OMF TolC absolutely required the respective tip-regions. The interaction of DevB with the IMF DevAC mainly involved the β-barrel and the lipoyl domain. Efficient binding to DevAC and TolC, substrate recognition and export activity by DevAC were dependent on stable DevB hexamers.     

Heavy metal transport by the CusCFBA efflux system

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi‐protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu⁺ and Ag⁺ ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function.     

An improved heuristic for haplotype inference

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.     

Gating at both ends and breathing in the middle: conformational dynamics of TolC

Drug extrusion via efflux through a tripartite complex (an inner membrane pump, an outer membrane protein, and a periplasmic protein) is a widely used mechanism in Gram-negative bacteria. The outer membrane protein (TolC in Escherichia coli; OprM in Pseudomonas aeruginosa) forms a tunnel-like pore through the periplasmic space and the outer membrane. Molecular dynamics simulations of TolC have been performed, and are compared to simulations of Y362F/R367S mutant, and to simulations of its homolog OprM. The results reveal a complex pattern of conformation dynamics in the TolC protein. Two putative gate regions, located at either end of the protein, can be distinguished. These regions are the extracellular loops and the mouth of the periplasmic domain, respectively. The periplasmic gate has been implicated in the conformational changes leading from the closed x-ray structure to a proposed open state of TolC. Between the two gates, a peristaltic motion of the periplasmic domain is observed, which may facilitate transport of the solutes from one end of the tunnel to the other. The motions observed in the atomistic simulations are also seen in coarse-grained simulations in which the protein tertiary structure is represented by an elastic network model.     

Structure of an atypical periplasmic adaptor from a multidrug efflux pump of the spirochete Borrelia burgdorferi

Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35 Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.     

Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of Gram-negative bacteria

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