Elucidation of the structure and molecular mechanism of the tripartite multidrug efflux pumps in the Gram-negative pathogens: Vibrio cholerae and Neisseria gonorrhoeae

In bacteria, multidrug efflux systems have been identified as significant determinants of resistance recently. These resistance pumps are widely distributed in bacterial species and many pathogenic bacteria posses them, which play an important role in their intrinsic and acquired multidrug resistances. The RND and MATE family transporters have also been shown to be involved in the pathogenicity of bacteria. Knowledge of the structure and mechanism of these transporter proteins would be exceedingly useful in the design of inhibitors. In Gram-negative bacteria, multidrug resistance is conferred in part by the tripartite multidrug efflux pumps that are composed of an inner membrane transport protein, a membrane fusion protein and an outer membrane protein. One such tripartite pump, VceCAB of Vibrio cholerae, is composed of an inner membrane H(^+)-antiporter VceB, a membrane fusion protein VceA and an outer membrane channel VceC. To investigate the role of this pump in the multidrug resistance of Vibrio cholerae, we have characterized functionally and structurally the three components of the VceCAB pump and the regulator VceR. The crystal structure of VceC was determined at 1.8 Â resolutions. Despite the very low degree of sequence identity between them, VceC shares the same overall architecture as TolC, consisting of three domains: the ß-domain, the α-domain and the equatorial domain. The trimeric VceC packs in laminar sheets in the crystal that resemble membranes. Like TolC, the α-barrel of the VceC channel at the periplasmic end is closed through the packing interactions of coiled-coil helices, but the residues that maintain the closed state of the channels of VceC and TolC are different. The ß-barrel region of VceC is also closed, whereas the ß-barrel region of TolC is open to the extracellular medium. The channel interior of VceC is generally electronegative and contains two rings of clusters negative charge. The ring made by residues Glu(^397) and Glu(^303) is conserved in OprM, but is not in TolC. Mutagenesis assay of this negative charged ring indicated its functional role during transport. The optimal desolvation area (ODA) on the surface of VceC is different from that of TolC, suggesting distinct architectures of VceC-based and TolC-based tripartite pumps. Sub-cellular fractionation of cells expressing full length and truncated VceA suggested that VceA is anchored to the IM via a transmembrane helix. Analytical gel filtration chromatography experiments revealed that the periplasmic domain of VceA that was expressed in the periplasm of E.coil forais a trimer, which could represent its oligomeric state in the VceCAB pump. The three components of tripartite pumps are easy to dissociate in vitro, making it difficult to co-crystallize them. We overproduced the protein complex in which VceA (12-406) is in complex with the VceB-VceA fusion protein. This complex was stable during purification, which could provide an invaluable way for co-crystallization of these two components of the VceCAB pump. An analytical gel filtration and DLS experiments indicated that the basic functional unit of VceR is a dimer; the binding of substrate СССР to VceR has been determined to occur with a Hill coefficient of about four, and thus each VceR dimer binds four СССР molecules. This stoichiometry of drug/VceR-subunit is different from that of other transcriptional regulators in the TetR/CamR family. There are differences between MtrD and other RND family multidrug efflux pumps. The knowledge of difference will be important for understanding the mechanism of these family transporters. In this study, we successfully overexpressed, purified and crystallized MtrD. The resolution of MtrD crystals was optimised to 7-10 Å at present

Structure and mechanism of bacterial tripartite efflux pumps.

Efflux pumps are membrane proteins which contribute to multi-drug resistance. In Gram-negative bacteria, some of these pumps form complex tripartite assemblies in association with an outer membrane channel and a periplasmic membrane fusion protein. These tripartite machineries span both membranes and the periplasmic space, and they extrude from the bacterium chemically diverse toxic substrates. In this chapter, we summarise current understanding of the structural architecture, functionality, and regulation of tripartite multi-drug efflux assemblies.ER

Structure, mechanism and cooperation of bacterial multidrug transporters.

Cells from all domains of life encode energy-dependent trans-membrane transporters that can expel harmful substances including clinically applied therapeutic agents. As a collective body, these transporters perform as a super-system that confers tolerance to an enormous range of harmful compounds and consequently aid survival in hazardous environments. In the Gram-negative bacteria, some of these transporters serve as energy-transducing components of tripartite assemblies that actively efflux drugs and other harmful compounds, as well as deliver virulence agents across the entire cell envelope. We draw together recent structural and functional data to present the current models for the transport mechanisms for the main classes of multi-drug transporters and their higher-order assemblies.BL and DD are supported by the Medical Research Council (MRC), Human Frontiers Science Program (HFSP), and the Wellcome Trust. Work in the Van Veen lab is supported by the Biotechnology and Biological Sciences Research Council (BBSRC), MRC, HFSP, Royal Society, Society for Antimicrobial Chemotherapy (BSAC), Herchel Smith Foundation, and Commonwealth Trust. Work in the Pos lab is supported by the German Research Foundation (SFB 807, Transport and Communication across Biological Membranes and FOR2251, Adaptation and persistence of the emerging pathogen Acinetobacter baumannii), the DFG-EXC115 (Cluster of Excellence Macromolecular Complexes at the Goethe-University Frankfurt), Innovative Medicines Initiative Joint Undertaking Project Translocation (IMI-Translocation), EU Marie Curie Actions ITN, HFSP and the German-Israeli Foundation (GIF). The SM laboratory is supported by ERATO Murata Lipid Active Structure Project, Japan Science and Technology Agency, the Advanced Research for Medical Products Mining Program of the National Institute of Biomedical Innovation (NIBIO) and HFSP.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.sbi.2015.07.01

Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis.

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5.

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.Open Access funding enabled and organized by Projekt DEAL

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC

Abstract: Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors

Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment.

The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.ER

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing

Multidrug efflux pumps:structure, function and regulation

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC

Abstract: Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors