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BBA - Proteins and Proteomics (v.1794, #5)

Editorial Board (pp. i).

Guest editor introduction by Helen I. Zgurskaya (pp. 723-724).

Molecular basis of multidrug transport by ABC transporters by Markus A. Seeger; Hendrik W. van Veen ⁎ (pp. 725-737).

Multidrug ABC transporters such as the human multidrug resistance P-glycoprotein (ABCB1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. These transport systems contain two nucleotide-binding domains (NBDs) where ATP is bound and hydrolyzed and two membrane domains (MDs) which mediate vectorial transport of substrates across the cell membrane. Recent crystal structures of the bacterial ABCB1 homologues Sav1866 from Staphylococcus aureus and MsbA from Salmonella typhimurium and other organisms shed light on the possible conformational states adopted by multidrug ABC transporters during transport. These structures help to interpret cellular and biochemical data gathered on these transport proteins over the past three decades. However, there are contradictory views on how the catalytic cycle of ATP binding and hydrolysis by the NBDs is linked to the change in drug binding affinity at the MDs, which underlies the capture (high affinity) of the transported drug on one side of the membrane and its release (low affinity) on the other. This review provides an overview of the current evidence for the different transport models and establishes the most recent structure-function relationships in multidrug ABC transporters.

Keywords: ATP-binding cassette transporter; Catalytic cycle; Energy-coupling; Multidrug binding and translocation; Structure-function relationships

Bacterial multidrug transport through the lens of the major facilitator superfamily by Nir Fluman; Eitan Bibi (pp. 738-747).

Multidrug transporters are membrane proteins that expel a wide spectrum of cytotoxic compounds from the cell. Through this function, they render cells resistant to multiple drugs. These transporters are found in many different families of transport proteins, of which the largest is the major facilitator superfamily. Multidrug transporters from this family are highly represented in bacteria and studies of them have provided important insight into the mechanism underlying multidrug transport. This review summarizes the work carried out on these interesting proteins and underscores the differences and similarities to other transport systems.

Keywords: Secondary transport; Multidrug transport; Multidrug resistance; MDR; Major facilitator superfamily; MFS

EmrE, a model for studying evolution and mechanism of ion-coupled transporters by Shimon Schuldiner (pp. 748-762).

EmrE is a small (110 residues) SMR transporter from Escherichia coli that extrudes positively charged aromatic drugs in exchange for two protons, thus rendering bacteria resistant to a variety of toxic compounds. Due to its size, stability and retention of its function upon solubilization in detergent, EmrE provides a unique experimental paradigm for the biochemical and biophysical studies of membrane based ion-coupled transporters. In addition, EmrE has been in center stage in the past two years because it provides also a paradigm for the study of the evolution of membrane proteins. Controversy around this topic is still going on and some novel concepts are surfacing that may contribute to our understanding of evolution of topology of membrane proteins. Furthermore, based on the findings that the cell multidrug transporters interact functionally we introduce the concept of a cell Resistosome.

Keywords: Multidrug resistance; Ion-coupled transport; Membrane protein; Topology; SMR transporter; Oligomeric structure; Resistosome

Multidrug efflux transporters in the MATE family by Teruo Kuroda; Tomofusa Tsuchiya (pp. 763-768).

The MATE ( Multidrug And Toxic Compound Extrusion) family is the most recently categorized one among five multidrug efflux transporter families. As far as we know, about twenty MATE transporters have been characterized so far. According to the information in sequence databases, huge numbers of MATE transporters seem to be present in various microorganisms. In this review, we would like to summarize the properties of the MATE-family transporters.

Keywords: Multidrug efflux transporter; MATE

Mechanisms of RND multidrug efflux pumps by Hiroshi Nikaido; Yumiko Takatsuka (pp. 769-781).

RND (Resistance-Nodulation-Division) family transporters are widespread especially among Gram-negative bacteria, and catalyze the active efflux of many antibiotics and chemotherapeutic agents. They have very large periplasmic domains, and form tripartite complexes with outer membrane channels and periplasmic adaptor proteins. AcrAB–TolC complex of Escherichia coli, which pumps out a very wide range of drugs, has been studied most intensively. Early studies showed that the transporter captures even those substrates that cannot permeate across the cytoplasmic membrane, such as dianionic β-lactams, suggesting that the capture can occur from the periplasm. It was also suggested that the capture occurs from the cytoplasmic membrane/periplasm interface, because most substrates contain a sizable hydrophobic domain; however, this may simply be a reflection of the nature of the binding site within AcrB. Genetic studies of chimeric transporters showed that much of the substrate specificity is determined by their periplasmic domains. Biochemical studies with intact cells recently led to the determination of the kinetic constants of AcrB for some β-lactams, and the result confirms the old prediction that AcrB is a rather slow pump. Reconstitution of purified AcrB and its relatives showed that the pump is a drug/proton antiporter, that AcrA strongly stimulates the activity of the pump, and that AcrB seems to have a highest affinity for conjugated bile salts. Structural study with mutants of the network of charged residues in the transmembrane domain showed that protonation here produced a far-reaching conformational change, which was found to be present in one of the protomers in the asymmetric crystal structure of the wild-type AcrB. The functional rotatory hypothesis then predicts that the drug bound in the periplasmic domain is extruded through this conformational change initiated by the protonation of one of the residues in the aforementioned network, an idea that was recently supported by disulfide cross-linking as well as by the behavior of linked AcrB protomers.

Keywords: AcrB; AcrD; TolC; AcrA; Reconstitution; Disulfide cross-linking; Proton relay network

Drug transport mechanism of the AcrB efflux pump by Klaas M. Pos (pp. 782-793).

In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude cytotoxic substances from the cell directly into the medium bypassing periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump that extrudes multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB, a member of the Resistance Nodulation cell Division (RND) family, is the major site for substrate recognition and energy transduction of the entire tripartite system. The drug/proton antiport processes in this secondary transporter are suggested to be spatially separated, a feature frequently observed for primary transporters like membrane-bound ATPases. The recently elucidated asymmetric structure of the AcrB trimer reveals three different monomer conformations proposed to represent consecutive states in a directional transport cycle. Each monomer shows a distinct tunnel system with entrances located at the boundary of the outer leaflet of the inner membrane and the periplasm through the periplasmic porter (pore) domain towards the funnel of the trimer and TolC. In one monomer a hydrophobic pocket is present which has been shown to bind the AcrB substrates minocyclin and doxorubicin. The energy conversion from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (D407/D408/K940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel drug transport model combines the alternate access pump mechanism with the rotating site catalysis of F₁F_o ATPase as originally postulated by Jardetzky and Boyer, respectively, and suggests a working hypothesis for the transport mechanism of RND transporters in general.

Keywords: Abbreviations; Acr; acriflavine resistance; AcrB; acriflavine resistance protein B; L monomer; loose monomer (access monomer); T monomer; tight monomer (binding monomer); O monomer; open monomer (extrusion monomer); MDR; multidrug resistance; OM; Outer Membrane; IM; Inner Membrane; MIC; Minimal Inhibitory Concentration; RMSD; root mean square distance; PDB; Protein Data BankMacromolecular complex; Tripartite drug pump system; AcrB; RND; Membrane transporter; Drug efflux; Peristaltic mechanism; Alternate access; Drug transport; Antiport; Binding change mechanism

Structural and functional diversity of bacterial membrane fusion proteins by Helen I. Zgurskaya ⁎; Yoichi Yamada; Elena B. Tikhonova; Qiang Ge; Ganesh Krishnamoorthy (pp. 794-807).

Membrane Fusion Proteins (MFPs) are functional subunits of multi-component transporters that perform diverse physiological functions in both Gram-positive and Gram-negative bacteria. MFPs associate with transporters belonging to Resistance-Nodulation-cell Division (RND), ATP-Binding Cassette (ABC) and Major Facilitator (MF) superfamilies of proteins. Recent studies suggested that MFPs interact with substrates and play an active role in transport reactions. In addition, the MFP-dependent transporters from Gram-negative bacteria recruit the outer membrane channels to expel various substrates across the outer membrane into external medium. This review is focused on the diversity, structure and molecular mechanism of MFPs that function in multidrug efflux. Using phylogenetic approaches we analyzed diversity and representation of multidrug MFPs in sequenced bacterial genomes. In addition to previously characterized MFPs from Gram-negative bacteria, we identified MFPs that associate with RND-, MF- and ABC-type transporters in Gram-positive bacteria. Sequence analyses showed that MFPs vary significantly in size (200–650 amino acid residues) with some of them lacking the signature α-helical domain of multidrug MFPs. Furthermore, many transport operons contain two- or three genes encoding distinct MFPs. We further discuss the diversity of MFPs in the context of current views on the mechanism and structure of MFP-dependent transporters.

Keywords: Membrane fusion protein; Multidrug efflux; Membrane transport; Protein–protein interaction; Phylogenetic analysis

Outer membrane permeability and antibiotic resistance by Anne H. Delcour (pp. 808-816).

To date most antibiotics are targeted at intracellular processes, and must be able to penetrate the bacterial cell envelope. In particular, the outer membrane of gram-negative bacteria provides a formidable barrier that must be overcome. There are essentially two pathways that antibiotics can take through the outer membrane: a lipid-mediated pathway for hydrophobic antibiotics, and general diffusion porins for hydrophilic antibiotics. The lipid and protein compositions of the outer membrane have a strong impact on the sensitivity of bacteria to many types of antibiotics, and drug resistance involving modifications of these macromolecules is common. This review will describe the molecular mechanisms for permeation of antibiotics through the outer membrane, and the strategies that bacteria have deployed to resist antibiotics by modifications of these pathways.

Keywords: Outer membrane; Antibiotic; Porin; LPS; Resistance

Assembly and transport mechanism of tripartite drug efflux systems by Rajeev Misra ⁎; Vassiliy N. Bavro (pp. 817-825).

Multidrug efflux (MDR) pumps remove a variety of compounds from the cell into the external environment. There are five different classes of MDR pumps in bacteria, and quite often a single bacterial species expresses multiple classes of pumps. Although under normal circumstances MDR pumps confer low-level intrinsic resistance to drugs, the presence of drugs and mutations in regulatory genes lead to high level expression of MDR pumps that can pose problems with therapeutic treatments. This review focuses on the resistance nodulation cell division (RND)-class of MDR pumps that assemble from three proteins. Significant recent advancement in structural aspects of the three pump components has shed new light on the mechanism by which the tripartite efflux pumps extrude drugs. This new information will be critical in developing inhibitors against MDR pumps to improve the potency of prescribed drugs.

Keywords: Drug resistance; RND-type multidrug efflux pump; Tripartite pump assembly; Proton/drug antiporter; Membrane fusion protein; Outer membrane protein factor

Mechanisms of drug efflux and strategies to combat them: Challenging the efflux pump of Gram-negative bacteria by Jean-Marie Pagès; Leonard Amaral (pp. 826-833).

Chemoresistance presents a general health problem concerning the therapy of infectious disease and cancer. In this context, the worldwide dissemination of “multidrugresistant” (MDR) pathogens has severely reduced the efficacy of our antimicrobial weapons and dramatically increased the frequency of therapeutic failure. Because MDR bacterial infections involve the over-expression of efflux pumps that expel unrelated antibiotics before they can reach their targets, it is necessary to clearly define the molecular and genetic bases of the MDR mechanisms in order to combat these infectious diseases. This characterization of efflux pumps allows the definition of an original anti-resistance weapon, the efflux pump inhibitor (EPI). Several chemical families of EPIs have been now described and characterized. Among them several inhibitor compounds display an efficient activity and inhibit the major AcrAB-TolC and MexAB-OprM efflux systems which are the major efflux pumps responsible for MDR Gram negative clinical isolates. The use of these EPIs induces a significant reduction of resistance to one or more antibiotics to which these isolates were initially resistant. Hence, the EPI when used as an adjuvant to the given antibiotic, restores the activity of the antibiotic. The description of the responsible efflux mechanism at its structural and physiological level will make it possible to develop along intelligent lines an improved new generation of EPIs that can readily be added to the armamentarium of current and past “fallen by the wayside” antibiotic therapies.

Keywords: AcrAB-TolC; Antibiotic; Efflux pump; Chemosensitizer; Efflux pump inhibitor; Membrane channel; MexAB-OprM; Multidrug resistance; Gram negative bacteria; Transporter

Regulation and physiological function of multidrug efflux pumps in Escherichia coli and Salmonella by Kunihiko Nishino; Eiji Nikaido; Akihito Yamaguchi (pp. 834-843).

Multidrug efflux is an obstacle to the successful treatment of infectious diseases, and it is mediated by multidrug efflux pumps that recognize and export a broad spectrum of chemically dissimilar toxic compounds. Many bacterial genome sequences have been determined, allowing us to identify drug efflux genes encoded in the bacterial genome. Here, we present an approach to identifying drug efflux genes and their regulatory networks in Escherichia coli and Salmonella. Multidrug efflux pumps are often regulated by environmental signals and they are required for bacterial virulence in addition to multidrug resistance. It is now understood that these efflux pumps also have physiological roles. In this article, we investigate the physiological roles of drug efflux pumps in virulence. Because multidrug efflux pumps have roles in bacterial drug resistance and virulence, we propose that drug efflux pumps have greater clinical relevance than previously considered.

Keywords: Drug efflux pump; Regulation; Virulence; Multidrug resistance; Two-component signal transduction system; Escherichia coli; Salmonella; CpxA/CpxR; BaeS/BaeR; EvgS/EvgA; AcrS; RamA; HNS; AcrAB; MacAB; MdsABC

Structures of AcrR and CmeR: Insight into the mechanisms of transcriptional repression and multi-drug recognition in the TetR family of regulators by Mathew D. Routh; Chih-Chia Su; Qijing Zhang; Edward W. Yu ⁎ (pp. 844-851).

The transcriptional regulators of the TetR family act as chemical sensors to monitor the cellular environment in many bacterial species. To perform this function, members of the TetR family harbor a diverse ligand-binding domain capable of recognizing the same series of compounds as the transporters they regulate. Many of the regulators can be induced by a wide array of structurally unrelated compounds. Binding of these structurally unrelated ligands to the regulator results in a conformational change that is transmitted to the DNA-binding region, causing the repressor to lose its DNA-binding capacity and allowing for the initiation of transcription. The multi-drug binding proteins AcrR of Escherichia coli and CmeR from Campylobacter jejuni are members of the TetR family of transcriptional repressors that regulate the expression of the multidrug resistant efflux pumps AcrAB and CmeABC, respectively. To gain insights into the mechanisms of transcriptional regulation and how multiple ligands induce the same physiological response, we determined the crystal structures of the AcrR and CmeR regulatory proteins. In this review, we will summarize the new findings with AcrR and CmeR, and discuss the novel features of these two proteins in comparison with other regulators in the TetR family.

Keywords: TetR family; Escherichia coli; AcrR; Campylobacter jejuni; CmeR; Transcriptional regulator; Multidrug resistance

Coordinate control of lipid composition and drug transport activities is required for normal multidrug resistance in fungi by Puja Shahi; W. Scott Moye-Rowley (pp. 852-859).

Pathogenic fungi present a special problem in the clinic as the range of drugs that can be used to treat these types of infections is limited. This situation is further complicated by the presence of robust inducible gene networks encoding different proteins that confer tolerance to many available antifungal drugs. The transcriptional control of these multidrug resistance systems in several key fungi will be discussed. Experiments in the non-pathogenic Saccharomyces cerevisiae have provided much of our current understanding of the molecular framework on which fungal multidrug resistance is built. More recent studies on the important pathogenic Candida species, Candida albicans and Candida glabrata, have provided new insights into the organization of the multidrug resistance systems in these organisms. We will compare the circuitry of multidrug resistance networks in these three organisms and suggest that, in addition to the well-accepted drug efflux activities, the regulation of membrane composition by multidrug resistance proteins provides an important contribution to the resistant phenotypes observed.

Keywords: Multidrug resistance; Transcription; ABC transporter; Candida albicans; Candida glabrata; Saccharomyces cerevisiae; Sphingolipid; Phospholipid asymmetry

A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function by King Leung Fung; Michael M. Gottesman ⁎ (pp. 860-871).

The MDR1 ( ABCB1) gene encodes a membrane-bound transporter that actively effluxes a wide range of compounds from cells. The overexpression of MDR1 by multidrug-resistant cancer cells is a serious impediment to chemotherapy. MDR1 is expressed in various tissues to protect them from the adverse effect of toxins. The pharmacokinetics of drugs that are also MDR1 substrates also influence disease outcome and treatment efficacy. Although MDR1 is a well-conserved gene, there is increasing evidence that its polymorphisms affect substrate specificity. Three single nucleotide polymorphisms (SNPs) occur frequently and have strong linkage, creating a common haplotype at positions 1236C>T (G412G), 2677G>T (A893S) and 3435C>T (I1145I). The frequency of the synonymous 3435C>T polymorphism has been shown to vary significantly according to ethnicity. Existing literature suggests that the haplotype plays a role in response to drugs and disease susceptibility. This review summarizes recent findings on the 3435C>T polymorphism of MDR1 and the haplotype to which it belongs. A possible molecular mechanism of action by ribosome stalling that can change protein structure and function by altering protein folding is discussed.

Keywords: Abbreviations; ABC; ATP-binding cassette; ATP; adenosine triphosphate; ADP; adenosine diphosphate; CFTR; cystic fibrosis trans-membrane conductance regulator; CYP3A4; cytochrome P450 3A4; CYP3A5; cytochrome P450 3A5; IVS; intervening sequence; MDR1; multidrug resistant 1; SNP; single nucleotide polymorphism; TM; trans-membrane; tRNA; transfer RNA; UTR; untranslated regionABCB1; MDR1; Polymorphism; Haplotype; Ribosome stalling

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BBA - Proteins and Proteomics (v.1794, #5)