Multidrug ABC transporters in bacteria

Research in Microbiology xxx (xxxx) xxx

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Multidrug ABC transporters in bacteria dric Orelle*, Khadija Mathieu, Jean-Michel Jault** Ce Universit e de Lyon, CNRS, UMR5086 “Molecular Biology and Structural Biochemistry”, IBCP, 7 passage du Vercors, 69367 Lyon, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 May 2019 Accepted 17 June 2019 Available online xxx

Multidrug efflux transporters are a plague in the antibiotic resistance mechanisms as they confer the capacity of bacteria to evade most of current therapies. Although these transporters were initially discovered as proton-motive driven pumps, another class of multidrug efflux transporters has emerged in the mid-90s that are powered by ATP hydrolysis. This new class of transporters belongs to one of the largest families of proteins, the ATP-Binding Cassette (ABC) transporters, which are involved in the influx or efflux of a huge variety of molecules. Tremendous progresses have been made in the recent years regarding the functioning mechanism of multidrug efflux ABC transporters, in particular with the accumulation of 3D structures, but many questions remain unsolved. In this review, we will give an overview of our current knowledge on the structure and function of multidrug ABC transporters with an emphasis on bacterial pumps. © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: ABC transporter Antibiotic resistance Multidrug resistance Efflux pumps

Multidrug efflux pumps from various families can cause antibiotic resistance in bacteria [1]. Induction of several of these pumps, although conferring a low level of resistance, is often the first step to drug resistance that eventually leads to higher level of resistance by acquiring chromosomal mutations for antibiotic targets [2e4]. One category of multidrug efflux pumps uses the energy of ATP hydrolysis to expel drugs and they belong to the very large superfamily of ATP-binding cassette (ABC) transporters [5]. The hallmark of ABC transporters is that they contain a similar topology with two transmembrane domains (TMD), which usually form the substrate translocation pathway, and two cytoplasmic nucleotide-binding domains (NBD) that energize the pump. These four domains can be borne on up to four separate polypeptides, or fused together in different combinations as exemplified in the archetype of multidrug efflux pumps, the eukaryotic P-glycoprotein (P-gp, ABCB1 or MDR1). In bacteria, multidrug efflux pumps often have one TMD fused to one NBD and they can form either homodimers or heterodimers.

1. Fold and conserved motifs of the nucleotide-binding domains (NBD) Many studies have been performed on isolated NBDs showing that these domains can independently bind ATP-Mg2þ or analogues * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C. Orelle), [email protected] (J.-M. Jault).

as inferred from the presence of all consensus motifs or conserved residues involved in ATP binding and hydrolysis. This was observed not only with isolated recombinant NBDs obtained from full-length or half-size transporters (e.g. P-gp [6], MRP [7], TAP1/TAP2 [8] or a related yeast transporter [9]) but also with NBDs from importers or from an ABC protein involved in FeeS cluster assembly, i.e. NBDs that stand alone as full polypeptides [10e12]. Solving the 3D structures for some of these domains confirmed their abilities to interact with nucleotides. This was exemplified by the first crystallized NBD, HisP, from the histidine importer of Salmonella typhimurium [13]. This ATP-binding subunit and subsequent other NBDs revealed that the fold of ABC transporters is rather well conserved with a L-shape structure made of three subdomains [12]. The first is a RecA-like subdomain present in many NTPases [14,15] and that carries the Walker-A (GX4GKT/S) and Walker-B (hy4D) motifs (Fig. 1A). Additional motifs are present in the RecAlike subdomain: (i) The Q-loop, a stretch of ~8 amino acids starting with a conserved glutamine and connecting the RecA-like and a-helical subdomains. A conformational switch of the glutamine residue during the catalytic cycle may be involved in transmitting conformational changes between NBDs and TMDs; (ii) The H-loop contains a conserved histidine that acts as a linchpin in ATP hydrolysis by interacting with the g-phosphate of ATP, the attacking water and the catalytic glutamate [16]; (iii) The D-loop is located downstream of the Walker B motif. It has a central role in the functionality of ABC transporters [17,18]. When the NBDs are sufficiently close to each other, the D-loops establish a complex

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and belongs to a loop located at the N-terminus of a a-helix. The role of this motif had remained elusive for a long time until the 3D structures of proper ABC dimers were solved (see NBD dimerization below). Another motif present in this subdomain is the X-loop, defined as TEVGERG sequence in Sav1866 [24]. It is only present in exporters and precedes the signature motif in the a-helical subdomain. Its name refers to the fact that it interacts and cross-talk with both intracellular domains (ICDs). Based on its proximity with the signature motif, it has been hypothesized to transmit conformational changes between the ATP binding-site and the TMDs [25e28] and this was recently demonstrated [29]. The other subdomain is called the b-subdomain. It contains the A-loop which is located upstream of the Walker A motif, and bears a conserved aromatic (A) residue that stacks against the adenine ring of the nucleotide, thereby stabilizing it [14,30]. 2. NBD dimerization is required for ATPase activity

Fig. 1. Conserved motifs found in the NBDs and the organization of the transient NBD dimer with the two nucleotide-binding sites (NBS) at the NBD interface. A, The A-loop (A), the Walker-A and -B motifs (WA and WB, respectively), the Q-loop, the X-loop, the putative catalytique glutamate adjacent to the Walker-B motif (E), the H-loop, the ABC signature or C-loop (C) and the D-loop are shown in color. Please note that the motifs shown above the sketch of the NBD participate in one nucleotide-binding site (NBS1) while the motifs shown below belong to the second nucleotide-binding site (NBS2). B, transient dimer organization of the two NBDs. In the case of homodimers (left panel), the two NBDs are identical and the two NBS located at the NBD interface are symmetrical. In the case of heterodimers (right panel), NBD1 and NBD2 are different and the two NBS are asymmetrical. Crippled motifs borne in NBD1, NBD2 or both (shown in italic with an asterik) create a degenerate NBS1. In contrast, normal motifs are present at the level of NBS2 creating a consensus site capable to hydrolyze nucleotides. Ad, adenine; Pi, phosphate. C, structure of the Sav1866 NBD dimer (PDB code: 2ONJ) drawn by Pymol. Two AMPPNP are shown in stick representation. The same color coding was used for the motifs in all panels. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

electrostatic and hydrogen bond network with the Walker A motif and H-loop of the opposite NBD, thereby allowing communication between the NBDs (see NBD dimerization below) [19]. Because of its position at the dimer interface, the ‘D-loop’ originally referred to ‘dimer’ [20] although it rather currently refers in the field to the invariant aspartic acid present in the motif. In addition, the D-loops also connect and stabilize the catalytic glutamate and attacking water [21,22]. Finally, the D-loop is also involved in the directionality and energetic of the transport [23]. The two other subdomains are specific to the ABC family. One is the a-helical subdomain, which contains the family signature motif, also known as the C-loop. Its sequence usually starts with LSGGQ

ATP binding to isolated NBDs usually occurs with a low affinity but induces notable structural changes within the domains [31]. Nevertheless, the ATPase activities that could be measured, if any, were often several orders of magnitude lower than in the whole transporters [6,7,11,32]. On the other hand, it was shown that NBDs could interact together to form a transient dimer that displayed a high ATPase activity with a positive cooperativity [33] (Fig. 1B and C). This was firm evidence suggesting that this transient dimer is the mandatory entity to get the full ATPase activity of the transporter. In support of this mechanism, it was shown that the rate of ATPase activity of the isolated NBD from the Mdl1p transporter is strictly dependent on its rate of dimerization [34]. Several lines of evidence endorsed the link between transient dimerization of the NBDs and the high ATPase activity. First and for most, the possibility to form stable NBD dimers upon mutations of essential residues that abrogated the ATPase activity. These ATPase inactive mutants were obtained by mutating the putative catalytic glutamate following the Walker-B motif in MJ0796, MJ1267 [33] or in the MBP-MalFGK2 transporter [35], or the histidine of the H-loop in the isolated NBD of HlyB [36]. Both mutants retained the ability to bind ATP that triggered the dimerization of the two NBDs. This resulted in a NBD dimer in a head-to-tail orientation that sandwiched two ATP molecules at their interface [16,37] (Fig. 1B and C). In fact, these structures revealed that the two ATP sites are shared between the two NBDs with one ATP site (site 1) formed by the A-loop, Q-loop, Walker A and B motifs and H-loop provided by the first NBD (NBD1) while the C- and D-loops are provided in trans by NBD2, and conversely for the second ATP site (site 2; Fig. 1B and C). The implication of the C-loop in the formation of a composite Nucleotide-Binding Site (NBS) at the interface of the two NBDs was also supported by mutagenesis [38e40] or NMR studies [7]. Regarding the NBD dimerization, structures of the MalK homodimers have been very informative too. Apart for the classical NBD structure harboring all the signatures found in every ABC transporter, MalK contains an additional C-terminal domain that maintain, at high protein concentrations, an interaction between two MalK subunits [41]. Therefore, it was possible to isolate dimers of MalK in the apo or nucleotide-bound states. In these two latter states, the NBD moieties of the two MalK subunits are physically disengaged, being maintained as a homodimer thanks to their additional C-terminal domains. In contrast, the ATP-bound state revealed that the two NBD moieties of MalK interact together in a head-to-tail conformation similar to those seen in other ABC transporters [41]. In addition, it was observed that in the presence of ATP and orthovanadate, leading to an inhibited state mimicking the transition state for ATP hydrolysis (i.e. an ADP-Mg-Vi trapped conformation), UV irradiation induced the photocleavage in both

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the Walker A and C motifs in the maltose transporter. This shows convincingly that ATP hydrolysis occurs only in the closed conformation, but it also reveals the proximity of the Walker A and C motifs in one ATP site. Because these motifs are too far apart in a single NBD, this emphasizes the need for a dimerization step forming two composite NBSs at the NBD interface [42]. While providing extremely valuable insights into the mechanism of ABC transporters, the structures of isolated NBDs sometimes led to misinterpretations of catalytic mechanisms since the TMDs reshape the geometry of the catalytic sites [22,43]. 3. Identification of multidrug ABC transporters in bacteria In the last two decades, many ABC transporters with drug transport abilities were identified (Table 1). It should be noted that in vivo resistance phenotypes are not easily observable due to the high redundancy of drug transporters (proton- or ATP-dependent) with overlapping specificities in bacterial genomes. For instance, Bacillus subtilis, Staphylococcus aureus and Escherichia coli may carry up to 30 putative drug efflux pumps [44,45]. Of note, besides the existence of classical drug efflux pumps, some ABC transporters often associated with two-component regulatory systems confer resistance to antimicrobial peptides (see for review [46]). Several methodologies have been employed to identify MDR transporters. In some cases, knocking out candidate genes resulted in increased drug susceptibilities, and heterologous overexpression of MDR transporters in sensitive strains increased drug resistance. Another successful and common strategy has been to perform transport experiments with inverted membrane vesicles overexpressing the transporters. Nevertheless, true physiological substrates of drug efflux pumps are usually not known. In the next two paragraphs, we will give information regarding the discovery and characterization of some multidrug ABC transporters. 3.1. Transporters with identical NBDs The first bacterial multidrug (MDR) ABC transporter was discovered in Lactococcus lactis and was named LmrA for Lactococcus multidrug resistance ATP [58]. LmrA functions as a homodimer [71]. Each monomer is made of one transmembrane domain containing 6 predicted transmembrane helices and one nucleotidebinding domain. The overexpression of LmrA in a drug-sensitive E. coli strain caused a resistance phenotype to several structurally unrelated compounds, i.e. ethidium, rhodamine 6 G, daunorubicin

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and TPPþ [58]. In addition, the accumulation of daunorubicin in inverted membrane vesicles was dependent on ATP hydrolysis and inhibited by reserpine, a classical inhibitor of drug efflux pumps. Energetics of LmrA was found to be quite unique with use of both ATP hydrolysis and ion antiporter capabilities [72]. Based on its homology with LmrA, an uncharacterized membrane protein from Bacillus subtilis was investigated [73]. Its functional overexpression in E. coli membranes [74] was used to show its ability to transport a variety of drugs (e.g. hoechst 33342, doxorubicin and 7-amino-actinomycin-D). This homodimeric transporter [75] was renamed BmrA (Bacillus multidrug resistance ATP) [48]. Interestingly, a Bacillus subtilis strain resistant to the antibiotic cervimycin C was isolated, in which a promoter mutation upregulated the expression of bmrA [47]. MacAB/TolC from E. coli was initially characterized as a macrolide-specific tripartite pump [53]. TolC is an outer membrane porin, while MacA is a Membrane Fusion Protein that interacts with both partner proteins [76]. Homologues are present in various Gram-negative bacteria [77,78]. MacB has an inverted topology: a N-terminal NBD is followed by a C-terminal TMD containing 4 transmembrane helices [52,77]. Nanomolar affinity interactions occur between TolC and MacA, and between MacA and MacB [79]. MacB is a dimer [80] whose ATPase activity is strongly stimulated by MacA [81]. Several drug-unrelated physiological functions have been proposed, including protoporphyrin [54] and enterotoxin STII export [52,55]. Recently, crystal structures of the pump illuminated its mechanism of action, and it was proposed that the assembled tripartite pump functions as a molecular bellows to propel substrates through the TolC exit channel, and the energy of the process is driven by MacB mechanotransmission [52]. Indeed, MacB does not transport substrates across the inner membrane, but instead couples ATP binding and hydrolysis with transmembrane conformational changes that are used to perform mechanical work in the extracellular space. Based on these findings, it was proposed that other ABC proteins might also use mechanotransmission to function [82]. DrrAB from Streptomyces peucetius exports daunorubicin and doxorubicin, two antibiotics used as anticancer agents, that this bacterium produces [83]. It was thought to be a narrow spectrum drug transporter until a biochemical characterization unraveled its ability to also transport the Hoechst 33342 and ethidium bromide [69]. Consistent with these observations, the homologous transporter in Mycobacterium tuberculosis was found to confer a resistance phenotype to several structurally unrelated drugs when expressed in M. smegmatis [62]. In contrast to LmrA and BmrA,

Table 1 Some bacterial ABC transporters implicated in multidrug efflux and examples of drug transported. Bacterium

Protein

Drugs transported

Selected references

Bacillus subtilis

BmrA BmrCD EfrAB EfrCD MacB HorA LmrA LmrCD Rv0194 Rv1218c DrrAB SmdAB AbcA VltAB PatAB DrrAB VcaM

Ethidium, Hoechst 33342, doxorubicin, 7-amino-actinomycin D, cervimycin C Hoechst 33342, BCECF-AM, mitoxantrone, doxorubicin Acriflavine, ethidium, DAPI, TPP, norfloxacin/ciprofloxacin, doxycycline Daunorubicin, doxorubicin, ethidium, Hoechst 33342, BCECF-AM Macrolides, bacitracin, colistin, enterotoxin STII, protoporphyrin Hop compounds, ethidium, Hoechst 33342 Ethidium, daunomycin, rhodamine 6G, TPPþ, macrolides, streptogramins, tetracyclines, chloramphenicol Daunomycin, Hoechst 33342, BCECF-AM, ethidium Ampicillin, chloramphenicol, streptomycin, tetracycline, vancomycin, erythromycin, novobiocin, ethidium Some pyridones, biaryl-piperazines, bisanilino-pyrimidines, pyrroles Doxorubicin, ethidium bromide, BCECF-AM norfloxacin, tetracycline, DAPI, Hoechst 33342 Beta-lactams, TPP, moenomycin, Phenol-soluble modulins Quaternary ammonium compounds (violagen, acriflavin, ethidium bromide, safranin) Berberine, acriflavin, ethidium, norfloxacin, Hoechst 33342 Daunorubicin, doxorubicin, Hoechst 33342, ethidium Tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunorubicine, DAPI, Hoechst 33342

[47,48] [49] [50] [51] [52e55] [56] [57,58] [59] [60] [61] [62] [63] [64e66] [67] [68] [69] [70]

Enterococcus faecalis Escherichia coli Lactobacillus brevis Lactococcus lactis Mycobacterium tuberculosis

Serratia marcescens Staphylococcus aureus Streptococcus mutants Streptococcus pneumoniae Streptococcus peucetius Vibrio cholerae

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where each monomer is a TMD fused to a NBD, DrrA is a single NBD subunit while DrrB is a TMD subunit predicted with 8 transmembrane helices [84]. Cross-linking experiments suggested a stoichiometry of 2 TMDs and 2 NBDs [85]. 3.2. Transporters with asymmetric NBDs Many multidrug ABC transporters have a degenerate ATPbinding site [86,87] in which the glutamate adjacent to the Walker B motif, the histidine of the H-loop and/or some residues in the signature motif of the opposite NBD are substituted by nonconsensual residues (Fig. 1B and C). Consequently, the functioning mechanism of these heterodimers is asymmetric with the degenerate NDB being poorly active in ATP hydrolysis [17,86]. LmrCD was shown to be a major multidrug resistance transporter in L. lactis [59,88,89] and its expression is under the control of a transcriptional repressor named LmrR [90]. Binding of drugs to LmrR reduces its affinity for LmrCD promoter thereby inducing the expression of the transporter [91]. BmrCD is a Bacillus subtilis transporter [49], and antibiotics targeting protein synthesis induce its expression through a ribosome-mediated transcriptional attenuation mechanism [92]. When overexpressed in E. coli membranes, BmrCD transports several drugs such as Hoechst 33342, doxorubicin or mitoxantrone [49]. The implication of PatAB in Streptococcus pneumoniae multidrug resistance was first demonstrated when inactivation of its genes increased the bacterium sensitivity to several drugs: ethidium bromide, berberine, acriflavine and norfloxacin [68]. After exposing a laboratory strain to ciprofloxacin, a multidrug resistant strain was isolated and found to upregulate the expression of PatA and PatB genes [93]. Later, such upregulation was also found in clinical isolates resistant to fluoroquinolones [94,95]. Several mechanisms were described for PatAB upregulation: promoter region and internal mutations [96], disruption of a transcriptional attenuator [97] or gene duplication [98]. Studies of the transporter overexpressed in E. coli showed that only the heterodimer was functional for drug efflux [99] and that PatAB strongly favors GTP as energy source to efflux drugs, especially at 37  C [100]. Interestingly, intracellular concentrations of ATP and GTP in Streptococcus pneumonia were both in mM ranges, and fluoroquinolone treatment substantially increased GTP concentrations [100]. EfrCD is a MDR transporter recently identified in Enterococcus faecalis [51]. When the genes encoding this transporter were inactivated, the mutated strain displayed increased sensitivity to several drugs, including Hoechst 33342, daunorubicin, doxorubicin and ethidium. Similar results were obtained when the transporter was heterogeneously overexpressed in a L. lactis strain hypersensitive to drugs due to the disruption of LmrA and LmrCD genes. 4. Architecture of drug transporters Drug exporters were crystallized in mainly two different conformational states: inward-facing and outward-facing (Fig. 2). An alternating access mechanism seems the most prevailing modus operandi in ABC transporters. It involves switching between two opposite conformations in which the substrate-binding site is alternatively accessible to the inner and the outer sides of the membrane [101e103]. Of note, a third occluded conformational state was also observed for McjD [104], PglK [105] and MsbA [106] that respectively transport microcin J25, lipid-linked oligosaccharides and lipid A. Substrate translocation is thus coordinated by the catalytic events, i.e. ATP binding, hydrolysis, and products release. Several studies on drug transporters suggest a lower affinity for

drugs in the outward-facing conformation thereby explaining their release outside the cell [71,107,108]. Type I exporters. Sav1866 [24,109] and TM287/288 [110] are respectively a homodimer and an asymmetric heterodimer. They were both described to have drug transport abilities. In these transporters and the archetypical P-glycoprotein [111], the two TMDs exhibit an extensive cross-over and each of them significantly contact the opposite NBD, notably through ICD2 (or either ICD2 or ICD4 in each moiety of the P-gp). In the inward-facing state, a central cavity is accessible from the cytoplasm. In the outwardfacing conformation, this cavity is shielded from the cytoplasm and the inner leaflet of the membrane, but accessible from the outer leaflet and the extracellular space. The transmembrane helices are connected via short extracellular loops and long intracellular loops that form the helical bundles. Consequently, the NBDs are relatively far from the membrane (~25 Å). The TMDs mainly contact the NBDs through the coupling helices of the intracellular domains ICD1 and ICD2. The coupling helix of ICD1 is located between TM helices 2 and 3 and interacts mostly with the NBD of its own monomer. The coupling helix of ICD2 located between TM helices 4 and 5 interacts only with the opposite monomer. This trans interaction of ICD2, as well as the distance of the NBDs from the membrane, are the hallmarks of the type I ABC exporters. Yet, the coupling helix 2 is rather similar to the coupling helix of the importers since it docks into a cleft or groove at the interface between the RecA-like and a-helical subdomains of the NBDs (Fig. 3). It should be noted that an alternative model to the classical alternating access mechanism has been proposed for the lipidlinked oligosaccharide flippase PglK [105]. Although the transporter can adopt inward- and outward-facing conformations, this study suggests that substrate directly binds the outward-facing state and is flipped upon ATP hydrolysis. Type II exporters and mechanotransmission transporters. Type II exporters were identified when the eukaryotic structures of ABCG5/G8 first and ABCG2 later were solved [112,113]. In these proteins, the NBDs are followed by the TMDs, and the global architecture is much closer to importers. There is no trans interaction of the TMDs, and the NBDs are close to the membrane. A similar architecture is seen with MacB, although the authors preferred to assign it to a different class since it was proposed to function via mechanotransmission, and not as a stricto sensu exporter [82]. 5. Drug-binding sites and polyspecificity The drug-binding site has been best characterized with P-gp. ABCB1 contains a large and promiscuous binding site, in which all transmembrane helices participate to substrate binding in the inward-facing conformation. The TMDs forms a translocation path in its center and the residues identified to be involved in substrate interactions are oriented towards an inner cavity of the TMDs and cluster halfway through the membrane (see recent reviews [114e116]). MDR transporters have the unique ability to recognize a wide variety of structurally unrelated substrates. The molecular basis for polyspecificity is not fully understood, but several interesting and plausible hypotheses have been proposed. The first is the existence of a large binding site that can accommodate multiples drugs. Shapiro and Ling demonstrated that P-gp could accommodate the simultaneous binding of at least two different drugs: Hoechst 33342 or colchicine at the so-called H site, and rhodamine 123 or anthracyclins at the R site. Indeed, transport with inverted membrane vesicles showed that low concentrations of a drug could reciprocally stimulate the transport of another drug [117]. This property is unique to MDR transporters and was also found for bacterial transporters such as LmrA [71] or BmrA [48].

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Fig. 2. 3D structures of selected exporters from the ABC family. The N-terminal half of P-glycoprotein (P-gp), TM287, ABCG5 and one monomer of McjD, Sav1866 and MacB are colored in green, while the C-terminal half of P-gp, TM288, ABCG8 and the other monomer of McjD, Sav1866 and MacB are shown in blue. When present, AMP-PNP is shown in red stick representation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Structural interface of the TMDs and NBDs in the type I exporter Sav1866 (PDB 2ONJ). A, one monomer is shown in green, while ICD2 and the coupling helix 2 (CH2) from the other monomer is colored in blue. The Q-loop is colored in orange, while the X-loop is shown in magenta. AMP-PNP is shown as red sticks. B, the X-loop of one monomer cross-talks with the coupling helices from the other monomer. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Drug binding studies also supported that DrrAB transporter contains multiple drug binding sites [118]. Moreover, several DrrAB mutants were identified that lost resistance to doxorubicin and verapamil simultaneously but retained resistance to Hoechst 33342 and/or ethidium, suggesting the existence of a drug binding site that can accommodate multiple drugs [119]. Interestingly, some transcriptional repressors of drug-efflux pumps also display this polyspecificity [91]. Their co-crystallization with different drugs illustrated the plasticity of a large multidrug-binding pocket that can accommodate several drugs, in some cases simultaneously [120,121]. Furthermore, based on a recent study with a MFS transporter from Candida albicans, the authors suggested that MDR transporters have an extended capacity brought by residues located at the periphery of a drug-binding core to accommodate compounds differing in size and structure [122]. The second explanation derives from the vacuum cleaner model that has been proposed for P-gp [123]. Due to their hydrophobicity and high membrane partition coefficient, drugs accumulate in the membrane up to several orders of magnitudes [124]. Polyspecificity can then be explained by high local substrate concentration that allows for low affinity and thus poorly-selective binding. Multidrug efflux pumps retrieve their substrates from the inner leaflet of the

membrane and then flip them into the outer leaflet or directly to the extracellular space. The third main explanation results from the flexibility of MDR transporters that can explore multiple states or conformations [125e128]. Conformational selection may provide a source of substrate promiscuity and the constant remodeling of the drugbinding sites may be an advantage to bind a large variety of drugs. For instance, multiple structures of P-gp showed significant movements of individual transmembrane helices, correlating with the opening and closing motion of the two halves of the protein. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a plausible mechanistic explanation for the polyspecificity of P-gp [129]. Finally, Ernst and collaborators have proposed a kinetic substrate selection model [130,131]. This model proposes that the time spent in the inward- or outward-facing states affects substrate selection and explains how two substrates with identical affinities, but dissimilar kon and koff, may be transported with different efficiencies. If the ATPase activities of these transporters were tightly coupled to the drug translocation process, then only the substrates capable of stimulating the ATPase activity would be transported, and a tight coupling might only be achieved at the cost of substrate

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diversity. The high basal ATPase activity of multidrug ABC allows sampling multiple states in a fast process. Being able to rapidly switch between the inward- and outward-conformations may help drugs to be rapidly expelled out of the cell before unbinding from the transporter in an on and off process, especially given the relatively low affinity for many drugs. Although MDR transporters are promiscuous in their drug transport capabilities, it would be naive and inaccurate to envision a lack of selectivity. Most bacterial MDR transporters from the ABC family fail to confer resistance to many drugs [51,88] and relatively small variations in chemical structures may also strongly alter drug transport abilities. For instance, PatAB induces resistance to some fluoroquinolones but not to others [68]. This may be one of the reasons explaining the high number of drug transporters with various specificities in bacterial genomes. 6. ATPase activity and transport mechanism Multidrug transporters typically display a high basal ATPase activity, which for bacterial transporters is often moderately stimulated by drugs [48,51,132]. However, several lines of evidence suggest that drugs binding facilitate the dimerization of the nucleotide-binding domains thereby stimulating ATP hydrolysis [133,134]. An essential question regarding the catalytic mechanism of ABC transporter is to know which is(are) the residue(s) responsible for the ATPase activity of ABC transporters. Two extreme scenarios were envisioned including the substrate-assisted catalysis or a general base catalysis [16]. Based on structure comparison between different families of ATPases bearing a recA-like domain, it was initially proposed that the invariant Glu residue following the Walker-B motif is involved in the proton abstraction from a catalytic water molecule [14,135]. The later mechanism was supported by crystallographic studies showing that this glutamate residue seemed to be ideally positioned to abstract a proton from a catalytic water molecule [10,13,22]. Mutagenesis and kinetics studies substantiated the direct role of this Glu residue in the ATPase activity of ABC transporters [33], and its replacement by several other residues in BmrA (e.g. Asp, Gln, Ala…) invariably led to a transporter devoid of ATPase activity [136]. More recently, molecular dynamics simulation performed on the ButCD-F vitamin B12 importer further supported the role of this Glu residue as the catalytic base of the ATPase reaction [137]. On the other hand, molecular dynamics simulation performed on the whole maltose transporter suggested that this invariant Glu, as well as the conserved His, act in concert to stabilize the transition state [138], and this study was therefore in favor of a substrate-assisted catalysis. Also, the isolated NBD from HlyB with a mutation of the corresponding Glu residue (E631Q) still retained ~ 10% of the ATPase activity of the wild-type subunit and this argued against the involvement of this residue as a catalytic base [16]. This, together with the lack of effect of D2O on the ATPase activity of the HlyB NBD [16], lent support to a substrate-assisted catalysis, as also pointed out by molecular dynamics simulation on the same NBD [139]. However, the work of Oldham and Chen emphasized that the geometry of the NBS is finely tuned by the interaction with the TMD, and this has a strong impact notably on the proper orientation of the Glu residue in the active site [22]. Possibly related to this, the conserved Glu residue of the SufC NBD is located in a very unusual 310 helix; this peculiar conformation flips its side-chain out of the ATP-binding site and this domain displays an extremely low basal ATPase activity (kcat ~ 1.2 min1) [11]. Likewise, an unusual salt bridge was reported in the HlyB NBD between the invariant Lys residue of the Walker-A motif and the conserved Glu residue thus precluding the binding of ATP [140]. This latent conformation was proposed to be a way to regulate the

ATPase activity of HlyB. Overall, despite the profound knowledge gained from studies on isolated NBDs, unraveling the detailed molecular bases of ATPase activity using these domains alone, without the TMD, might lead to debatable conclusions. In particular, this raises some reservations about the relevance of doing molecular dynamics simulation on isolated NBDs alone [139]. For multidrug ABC transporters, the stoichiometry of ATP molecules hydrolyzed per allocrite transported is a puzzling question difficult to tackle due to the amphiphilic nature of the drugs [141,142]. These molecules spontaneously partition into lipid bilayers where they bind to multidrug transporters and are effluxed in an ATP-dependent manner [143]. Yet, they concomitantly tend to diffuse passively through the membrane, and this diffusion makes the precise determination of active transport hard to measure accurately. Moreover, multidrug ABC exporters exhibit an inherent basal ATPase activity in the absence of transported molecules [48,51,74,100,132]. It is likely that this idiosyncratic ‘uncoupled’ activity might contribute to some extent to the overall ATPase activity even when drugs are present, i.e. not all multidrug exporters will be saturated by the added drug at any given time and ‘empty’ transporters could contribute significantly to ATP hydrolysis even if drugs are present at high concentrations. This could be misleading especially for multidrug transporters whose basal ATPase activity is already very high and/or if their basal ATPase is poorly stimulated by drug addition (e.g. [48]). These caveats might explain the differences found for drug export by the P-gp, with a stoichiometry varying from 1 to 2.8 ATP molecule hydrolyzed per drug transported [143e145]. It should be noted that different allocrites were used in these experiments that might possibly impact the ATP/drug ratio (i.e. the transport of two different drugs could require a different number of ATP hydrolyzed). In the case of multidrug exporters with an asymmetric functioning mechanism, the degenerate site is unlikely to hydrolyze ATP and one would expect an ATP/ drug ratio close to 1. Regarding other ABC transporters such as the peptide exporter TAP [146] or type I ABC importers [147,148], their ATPase activity is strictly coupled to allocrite transport. For type I importers like MalFGK2 or OpuA, it seems that the ATP/allocrite ratio is close to 2 [149,150]. Intriguingly, type II importers such as BtuCDF have a high basal ATPase activity and they show an apparent stoichiometry of ~100 ATP/vitamin B12 transported [151]. This ‘lousy’ coupling efficiency was proposed to be related to the availability of, and requirement for, molecules transported, i.e. scarce molecules with a reduced need [152]. A variable ATP/allocrite stoichiometry is presumably the corollary of the diversification in structure and function among ABC transporters and illustrates once again the variation on a common theme found in this family. Still, this issue has consequences for the catalytic cycle of ABC transporters and in particular for multidrug transporters. During the catalytic cycle, the two NBDs of ABC transporters engage and disengage with each other, although the degree of opening is also dependent on the lipid environment [153]. Because the two ATP-binding sites are localized at the interface of the two monomers, ATP binding promotes the formation of a closed conformation [33,154]. The release of Pi and/or ADP destabilizes the dimer such that the NBDs move apart from each other. In addition to the interdomain movement, the RecA-like and a-helical subdomains within each NBD rotate toward each other upon ATP binding and move outward in the post-hydrolysis stage [12,155]. Hence, the energy of ATP binding and hydrolysis is coupled to conformational changes in the TMD thereby mediating alternating access of the substrate-binding site to each side of the membrane. Several mechanistic models of drug transport have been proposed [116]. The two Nucleotide-Binding Sites (NBS) of P-gp were shown quite early on by Senior and his colleagues to be catalytically active as the trapping of Mg-ADP-Vi occurs with a similar efficiency in

Please cite this article as: C. Orelle et al., Multidrug ABC transporters in bacteria, Research in Microbiology, https://doi.org/10.1016/ j.resmic.2019.06.001

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either site [156]. In line with this, isolated membranes containing each half of human P-gp expressed separately showed similar levels of ATPase activity [157]. On the other hand, only one molecule of Mg-ADP-Vi (or ATP analogue) could be trapped, as also observed with the maltose transporter or homodimeric BmrA [136,158], and this was sufficient to prevent ATP hydrolysis by P-gp [159,160]. This showed that the two ATP sites cannot work simultaneously. The alternating catalytic cycle was thus proposed for P-gp where in the presence of two bound ATP, hydrolysis occurs at a single ATP site and this will promote drug translocation [161]. This model was later refined with the presence of the occluded state for nucleotides, i.e. two ATP bound in a closed conformation of the NBDs but one with higher affinity that is committed to ATP hydrolysis [162]. The presence of one ATP molecule in a more occluded conformation was supported by molecular dynamic studies performed on BtuCD [163] and also by the structure of the NBD of HlyB. This shows an asymmetry of the two ATP-binding sites in the presence of Mg2þ and in particular the opening of a phosphate exit tunnel in one of the two sites [164]. An asymmetry was also revealed by molecular dynamic simulations starting with the seemingly symmetric Sav1866 structure, and in particular at the level of the X-loop [28,165]. Besides, it was shown for P-gp that hydrolysis of one ATP molecule considerably reduces the affinity for a photoactivable drug analogue, suggesting that this step is sufficient for drug efflux. Then a second molecule of ATP was required to reset the transporter in a conformation competent for drug binding. This gave rise to a model where two ATP molecules are sequentially hydrolyzed per catalytic cycle, one for drug efflux and a second one to reset the transporter [166]. This requirement for ATP hydrolysis for drug efflux was challenged by the “ATP switch” model, which postulated that the NBD dimerization, promoted by ATP binding, is sufficient to export drugs, while hydrolysis of two ATP allows the resetting of the transporter [167]. To account for the ATPase activity of the NBD of Mdl1p, a processive clamp model was also proposed in which NBD dimerization induced by ATP binding is followed by hydrolysis of the two ATP molecules in a sequential manner before the transporter returns to the resting state [9]. Recently, a cryo-EM structure of P-glycoprotein, in which the catalytic glutamate mutation was introduced, was obtained in an outward-facing state in the presence of ATP [168], lending some support to these two models. The drug-binding cavity observed in previous inwardfacing structures was reorientated toward the extracellular space and remodeled to preclude substrate binding, suggesting that ATP binding, and not hydrolysis, promotes substrate release. Studies on BmrA in lipids suggested a large conformational change upon ATP binding [169]. Solid-state NMR suggested a flexible-to-rigid transition upon ATP binding and that the outward-facing conformation thereby induced is essentially similar to the one obtained by ATP hydrolysis and vanadate trapping [29]. Yet, whether ATP needs to be hydrolyzed in the ATP-bound conformation to somehow facilitate the release of the drugs still remains to be addressed. Molecular dynamic simulations on Sav1866 reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop to interfere with the transmembrane domain and favors its outward-open conformation [28]. When analysis was performed on TM287/288 and with longer timescales, however, the heterodimeric transporter underwent spontaneous conformational transitions from the inward-facing state via an occluded intermediate to an outward-facing state in response to ATP binding [170]. The recent publication of TM287/TM288 structure in the outwardfacing state supports this view, i.e. ATP binding alone is sufficient for the inward to outward conversion and presumably the active transport of one substrate molecule [171]. In addition, based on mutagenesis and the use of state-specific nanobodies, the authors

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suggested that subsequent closing of the extracellular gate is necessary to dissociate the NBD dimer after ATP hydrolysis at the consensus site, and to reset the transporter back to its inwardfacing state. Finally, a constant-contact model was proposed in which the NBDs remain partially associated during the catalytic cycle. ATP hydrolysis occurs alternately at each site. The site in which hydrolysis occurs is able to open and exchange ADP and Pi by an outward rotation of the RecA-like subdomain relative to the helical subdomain, while the other ATP-bound site remains closed [172].

7. Function of the degenerate nucleotide-binding domain As seen before, many ABC transporters are asymmetric with the presence of a degenerate NBS. The degenerate site is capable to bind nucleotides but has a low, if any, hydrolysis activity [17,86,87]. The functioning of ABC transporters with a degenerate site seems different from the classical one, since the presence of this peculiar site implies an asymmetric hydrolysis [173]. DEER (Double Electron Electron Resonance) experiments, that allows to measure distance between two nitroxide spin labels, or MD simulations were performed to understand how these transporters work. These observations have shown that for BmrCD, the conformational change from an « inward facing » to an « outward facing » requires ATP hydrolysis [174] unlike what is observed for TmrAB [175] or TM287/ TM288 [176], where nucleotide binding is sufficient to induce a conformational change. The functional role of the degenerate site remains poorly understood. Its role might be to prevent full separation of the NBDs as observed in homodimeric ABC exporters. The structural comparison of TM287/288 structures in the presence or absence of AMPPNP-Mg2þ suggested that nucleotide binding to the degenerate site prevents complete NBD separation. Importantly, the partial NBD contacts of inward-facing TM287/288 are mediated via the D-loops [19]. These D-loops are asymmetric and allow the allosteric coupling of the degenerate and consensus sites. These contacts are likely to facilitate NBD closure, because the NBDs remain appropriately aligned. By contrast, fully separated NBDs of homodimeric ABC exporters may sample a larger conformational space in their resting state and need to realign for their proper dimerization. Consequently, this pre-orientation in asymmetric transporters could facilitate NBD closure and may be relevant for substrate binding and regulatory mechanisms in these ABC exporters [176]. To better understand the implication of the degenerate site on the function of asymmetric ABC transporters, mutations in the degenerate motifs in Pdr5 were performed to restore a complete catalytic site [177]. However, the regenerated Pdr5 was severely impaired for drug transport and ATP hydrolysis. In a similar manner, mutations introduced in the degenerate site of EfrCD have lowered drug transport and ATP hydrolysis or have even inactivated ATPase activity with the mutation of the degenerate site Walker A lysine, indicating that ATP binding to the degenerate site is essential for EfrCD [17]. Overall, asymmetric transporter seems to function differently from classical ABC transporter with the need to keep a degenerate site which was conserved through evolution. These studies suggest that mutation in degenerate site might impact NBDeNBD or NBD-TMD communication and highlight the importance of the degenerate site in the function of the consensus one.

Conflict of interest The authors declare no of interest.

Please cite this article as: C. Orelle et al., Multidrug ABC transporters in bacteria, Research in Microbiology, https://doi.org/10.1016/ j.resmic.2019.06.001

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Acknowledgements The authors would like to thank the FINOVI Foundation (AO10 to CO and JMJ) and the Agence Nationale de la Recherche (ANR-17CE11-0045-01 to CO) for their financial support. KM is a grateful recipient of a 6-month PhD extension fellowship from the Fondadicale (FRM FDT201805005394). tion pour la Recherche Me

References [1] Chitsaz M, Brown MH. The role played by drug efflux pumps in bacterial multidrug resistance. Essays Biochem 2017;61:127e39. [2] El Meouche I, Dunlop MJ. Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation. Science (New York, N.Y) 2018;362:686e90. [3] Frimodt-Moller J, Rossi E, Haagensen JAJ, Falcone M, Molin S, Johansen HK. Mutations causing low level antibiotic resistance ensure bacterial survival in antibiotic-treated hosts. Sci Rep 2018;8:12512. [4] Schmalstieg AM, Srivastava S, Belkaya S, Deshpande D, Meek C, Leff R, et al. The antibiotic resistance arrow of time: efflux pump induction is a general first step in the evolution of mycobacterial drug resistance. Antimicrob Agents Chemother 2012;56:4806e15. [5] Lubelski J, Konings WN, Driessen AJ. Distribution and physiology of ABC-type transporters contributing to multidrug resistance in bacteria. Microbiol Mol Biol Rev 2007;71:463e76. [6] Dayan G, Baubichon-Cortay H, Jault JM, Cortay JC, Deleage G, Di Pietro A. Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein. Overexpression, purification, and role of cysteine 430. J Biol Chem 1996;271:11652e8. [7] Ramaen O, Sizun C, Pamlard O, Jacquet E, Lallemand JY. Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity. Biochem J 2005;391:481e90. [8] Gaudet R, Wiley DC. Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing. EMBO J 2001;20:4964e72. [9] Janas E, Hofacker M, Chen M, Gompf S, van der Does C, Tampe R. The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATPbinding cassette transporter Mdl1p. J Biol Chem 2003;278:26862e9. [10] Verdon G, Albers SV, Dijkstra BW, Driessen AJ, Thunnissen AM. Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotide-free and nucleotide-bound conformations. J Mol Biol 2003;330:343e58. [11] Watanabe S, Kita A, Miki K. Crystal structure of atypical cytoplasmic ABCATPase SufC from Thermus thermophilus HB8. J Mol Biol 2005;353:1043e54. [12] Karpowich N, Martsinkevich O, Millen L, Yuan YR, Dai PL, MacVey K, et al. Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter. Structure 2001;9: 571e86. [13] Hung LW, Wang IX, Nikaido K, Liu PQ, Ames GF, Kim SH. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 1998;396:703e7. [14] Geourjon C, Orelle C, Steinfels E, Blanchet C, Deleage G, Di Pietro A, et al. A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies. Trends Biochem Sci 2001;26:539e44. [15] Ye J, Osborne AR, Groll M, Rapoport TA. RecA-like motor ATPases–lessons from structures. Biochim Biophys Acta 2004;1659:1e18. [16] Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. Embo J 2005;24:1901e10. [17] Hurlimann LM, Hohl M, Seeger MA. Split tasks of asymmetric nucleotidebinding sites in the heterodimeric ABC exporter EfrCD. FEBS J 2017;284: 1672e87. [18] Schultz KM, Merten JA, Klug CS. Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA. Biochemistry 2011;50: 2594e602. [19] Hohl M, Hurlimann LM, Bohm S, Schoppe J, Grutter MG, Bordignon E, et al. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter. Proc Natl Acad Sci U S A 2014;111:11025e30. [20] Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, et al. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA doublestrand break repair and the ABC-ATPase superfamily. Cell 2000;101: 789e800. [21] Jones PM, George AM. Role of the D-loops in allosteric control of ATP hydrolysis in an ABC transporter. J Phys Chem A 2012;116:3004e13. [22] Oldham ML, Chen J. Snapshots of the maltose transporter during ATP hydrolysis. Proc Natl Acad Sci U S A 2011;108:15152e6. [23] Grossmann N, Vakkasoglu AS, Hulpke S, Abele R, Gaudet R, Tampe R. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter. Nat Commun 2014;5:5419. [24] Dawson RJ, Locher KP. Structure of a bacterial multidrug ABC transporter. Nature 2006;443:180e5.

[25] Becker JP, Van Bambeke F, Tulkens PM, Prevost M. Dynamics and structural changes induced by ATP binding in SAV1866, a bacterial ABC exporter. J Phys Chem B 2010;114:15948e57. [26] Damas JM, Oliveira AS, Baptista AM, Soares CM. Structural consequences of ATP hydrolysis on the ABC transporter NBD dimer: molecular dynamics studies of HlyB. Protein Sci 2011;20:1220e30. [27] Oancea G, O'Mara ML, Bennett WF, Tieleman DP, Abele R, Tampe R. Structural arrangement of the transmission interface in the antigen ABC transport complex TAP. Proc Natl Acad Sci U S A 2009;106:5551e6. [28] Xu Y, Seelig A, Berneche S. Unidirectional transport mechanism in an ATP dependent exporter. ACS Cent Sci 2017;3:250e8. [29] Lacabanne D, Orelle C, Lecoq L, Kunert B, Chuilon C, Wiegand T, et al. Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis. Commun Biol 2019;2:149. [30] Ambudkar SV, Kim IW, Xia D, Sauna ZE. The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett 2006;580:1049e55. [31] Hellmich UA, Monkemeyer L, Velamakanni S, van Veen HW, Glaubitz C. Effects of nucleotide binding to LmrA: a combined MAS-NMR and solution NMR study. Biochim Biophys Acta 2015;1848:3158e65. [32] Procko E, Ferrin-O'Connell I, Ng SL, Gaudet R. Distinct structural and functional properties of the ATPase sites in an asymmetric ABC transporter. Mol Cell 2006;24:51e62. [33] Moody JE, Millen L, Binns D, Hunt JF, Thomas PJ. Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters. J Biol Chem 2002;277:21111e4. [34] van der Does C, Presenti C, Schulze K, Dinkelaker S, Tampe R. Kinetics of the ATP hydrolysis cycle of the nucleotide-binding domain of Mdl1 studied by a novel site-specific labeling technique. J Biol Chem 2006;281:5694e701. [35] Oldham ML, Khare D, Quiocho FA, Davidson AL, Chen J. Crystal structure of a catalytic intermediate of the maltose transporter. Nature 2007;450:515e21. [36] Zaitseva J, Jenewein S, Wiedenmann A, Benabdelhak H, Holland IB, Schmitt L. Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter. Biochemistry 2005;44: 9680e90. [37] Smith PC, Karpowich N, Millen L, Moody JE, Rosen J, Thomas PJ, et al. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol Cell 2002;10:139e49. [38] Verdon G, Albers SV, van Oosterwijk N, Dijkstra BW, Driessen AJ, Thunnissen AM. Formation of the productive ATP-Mg2þ-bound dimer of GlcV, an ABC-ATPase from Sulfolobus solfataricus. J Mol Biol 2003;334: 255e67. [39] Buchaklian AH, Klug CS. Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA. Biochemistry 2006;45: 12539e46. [40] Tombline G, Bartholomew L, Gimi K, Tyndall GA, Senior AE. Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis. J Biol Chem 2004;279:5363e73. [41] Chen J, Lu G, Lin J, Davidson AL, Quiocho FA. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol Cell 2003;12: 651e61. [42] Fetsch EE, Davidson AL. Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter. Proc Natl Acad Sci U S A 2002;99:9685e90. [43] Bukowska MA, Hohl M, Geertsma ER, Hurlimann LM, Grutter MG, Seeger MA. A transporter motor taken apart: flexibility in the nucleotide binding domains of a heterodimeric ABC exporter. Biochemistry 2015;54: 3086e99. [44] Hassan KA, Skurray RA, Brown MH. Active export proteins mediating drug resistance in staphylococci. J Mol Microbiol Biotechnol 2007;12:180e96. [45] Paulsen IT, Chen J, Nelson KE, Saier Jr MH. Comparative genomics of microbial drug efflux systems. J Mol Microbiol Biotechnol 2001;3:145e50. [46] Gebhard S. ABC transporters of antimicrobial peptides in Firmicutes bacteria - phylogeny, function and regulation. Mol Microbiol 2012;86:1295e317. [47] Krugel H, Licht A, Biedermann G, Petzold A, Lassak J, Hupfer Y, et al. Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA. FEMS Microbiol Lett 2010;313:155e63. [48] Steinfels E, Orelle C, Fantino JR, Dalmas O, Rigaud JL, Denizot F, et al. Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis. Biochemistry 2004;43:7491e502. [49] Torres C, Galian C, Freiberg C, Fantino JR, Jault JM. The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter. Biochim Biophys Acta 2009;1788:615e22. [50] Lee EW, Huda MN, Kuroda T, Mizushima T, Tsuchiya T. EfrAB, an ABC multidrug efflux pump in Enterococcus faecalis. Antimicrob Agents Chemother 2003;47:3733e8. [51] Hurlimann LM, Corradi V, Hohl M, Bloemberg GV, Tieleman DP, Seeger MA. The heterodimeric ABC transporter EfrCD mediates multidrug efflux in Enterococcus faecalis. Antimicrob Agents Chemother 2016;60:5400e11. [52] Crow A, Greene NP, Kaplan E, Koronakis V. Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily. Proc Natl Acad Sci U S A 2017;114:12572e7. [53] Kobayashi N, Nishino K, Yamaguchi A. Novel macrolide-specific ABC-type efflux transporter in Escherichia coli. J Bacteriol 2001;183:5639e44.

Please cite this article as: C. Orelle et al., Multidrug ABC transporters in bacteria, Research in Microbiology, https://doi.org/10.1016/ j.resmic.2019.06.001

C. Orelle et al. / Research in Microbiology xxx (xxxx) xxx [54] Turlin E, Heuck G, Simoes Brandao MI, Szili N, Mellin JR, Lange N, et al. Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli. Microbiology Open 2014;3:849e59. [55] Yamanaka H, Kobayashi H, Takahashi E, Okamoto K. MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II. J Bacteriol 2008;190: 7693e8. [56] Sakamoto K, Margolles A, van Veen HW, Konings WN. Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA. J Bacteriol 2001;183:5371e5. [57] Putman M, Van Veen HW, Degener JE, Konings WN. Antibiotic resistance: era of the multidrug pump. Mol Microbiol 2000;36:772e3. [58] van Veen HW, Venema K, Bolhuis H, Oussenko I, Kok J, Poolman B, et al. Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1. Proc Natl Acad Sci U S A 1996;93:10668e72. [59] Lubelski J, Mazurkiewicz P, van Merkerk R, Konings WN, Driessen AJ. ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassettetype multidrug transporter. J Biol Chem 2004;279:34449e55. [60] Danilchanka O, Mailaender C, Niederweis M. Identification of a novel multidrug efflux pump of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2008;52:2503e11. [61] Balganesh M, Kuruppath S, Marcel N, Sharma S, Nair A, Sharma U. Rv1218c, an ABC transporter of Mycobacterium tuberculosis with implications in drug discovery. Antimicrob Agents Chemother 2010;54:5167e72. [62] Choudhuri BS, Bhakta S, Barik R, Basu J, Kundu M, Chakrabarti P. Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis. Biochem J 2002;367:279e85. [63] Matsuo T, Chen J, Minato Y, Ogawa W, Mizushima T, Kuroda T, et al. SmdAB, a heterodimeric ABC-Type multidrug efflux pump, in Serratia marcescens. J Bacteriol 2008;190:648e54. [64] Truong-Bolduc QC, Hooper DC. The transcriptional regulators NorG and MgrA modulate resistance to both quinolones and beta-lactams in Staphylococcus aureus. J Bacteriol 2007;189:2996e3005. [65] Villet RA, Truong-Bolduc QC, Wang Y, Estabrooks Z, Medeiros H, Hooper DC. Regulation of expression of abcA and its response to environmental conditions. J Bacteriol 2014;196:1532e9. [66] Yoshikai H, Kizaki H, Saito Y, Omae Y, Sekimizu K, Kaito C. Multidrugresistance transporter AbcA secretes Staphylococcus aureus cytolytic toxins. J Infect Dis 2016;213:295e304. [67] Biswas S, Biswas I. Role of VltAB, an ABC transporter complex, in viologen tolerance in Streptococcus mutans. Antimicrob Agents Chemother 2011;55: 1460e9. [68] Robertson GT, Doyle TB, Lynch AS. Use of an efflux-deficient streptococcus pneumoniae strain panel to identify ABC-class multidrug transporters involved in intrinsic resistance to antimicrobial agents. Antimicrob Agents Chemother 2005;49:4781e3. [69] Li W, Sharma M, Kaur P. The DrrAB efflux system of Streptomyces peucetius is a multidrug transporter of broad substrate specificity. J Biol Chem 2014;289:12633e46. [70] Huda N, Lee EW, Chen J, Morita Y, Kuroda T, Mizushima T, et al. Molecular cloning and characterization of an ABC multidrug efflux pump, VcaM, in Non-O1 Vibrio cholerae. Antimicrob Agents Chemother 2003;47:2413e7. [71] van Veen HW, Margolles A, Muller M, Higgins CF, Konings WN. The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism. EMBO J 2000;19:2503e14. [72] Agboh K, Lau CHF, Khoo YSK, Singh H, Raturi S, Nair AV, et al. Powering the ABC multidrug exporter LmrA: how nucleotides embrace the ion-motive force. Sci Adv 2018;4. eaas9365. [73] Steinfels E, Orelle C, Dalmas O, Penin F, Miroux B, Di Pietro A, et al. Highly efficient over-production in E. coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis. Biochim Biophys Acta 2002;1565: 1e5. [74] Mathieu K, Javed W, Vallet S, Lesterlin C, Candusso MP, Ding F, et al. Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain. Sci Rep 2019;9:2654. [75] Dalmas O, Do Cao MA, Lugo MR, Sharom FJ, Di Pietro A, Jault JM. Timeresolved fluorescence resonance energy transfer shows that the bacterial multidrug ABC half-transporter BmrA functions as a homodimer. Biochemistry 2005;44:4312e21. [76] Fitzpatrick AWP, Llabres S, Neuberger A, Blaza JN, Bai XC, Okada U, et al. Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump. Nat Microbiol 2017;2:17070. [77] Okada U, Yamashita E, Neuberger A, Morimoto M, van Veen HW, Murakami S. Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii. Nat Commun 2017;8:1336. [78] Rouquette-Loughlin CE, Balthazar JT, Shafer WM. Characterization of the MacA-MacB efflux system in Neisseria gonorrhoeae. J Antimicrob Chemother 2005;56:856e60. [79] Lu S, Zgurskaya HI. Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter. Mol Microbiol 2012;86:1132e43. [80] Lin HT, Bavro VN, Barrera NP, Frankish HM, Velamakanni S, van Veen HW, et al. MacB ABC transporter is a dimer whose ATPase activity and macrolidebinding capacity are regulated by the membrane fusion protein MacA. J Biol Chem 2009;284:1145e54.

9

[81] Tikhonova EB, Devroy VK, Lau SY, Zgurskaya HI. Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB. Mol Microbiol 2007;63:895e910. [82] Greene NP, Kaplan E, Crow A, Koronakis V. Antibiotic resistance mediated by the MacB ABC transporter family: a structural and functional perspective. Front Microbiol 2018;9:950. [83] Guilfoile PG, Hutchinson CR. A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin. Proc Natl Acad Sci U S A 1991;88:8553e7. [84] Gandlur SM, Wei L, Levine J, Russell J, Kaur P. Membrane topology of the DrrB protein of the doxorubicin transporter of Streptomyces peucetius. J Biol Chem 2004;279:27799e806. [85] Kaur P, Russell J. Biochemical coupling between the DrrA and DrrB proteins of the doxorubicin efflux pump of Streptomyces peucetius. J Biol Chem 1998;273:17933e9. [86] Lubelski J, van Merkerk R, Konings WN, Driessen AJ. Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric. Biochemistry 2006;45:648e56. [87] Zutz A, Hoffmann J, Hellmich UA, Glaubitz C, Ludwig B, Brutschy B, et al. Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus. J Biol Chem 2011;286:7104e15. [88] Lubelski J, de Jong A, van Merkerk R, Agustiandari H, Kuipers OP, Kok J, et al. LmrCD is a major multidrug resistance transporter in Lactococcus lactis. Mol Microbiol 2006;61:771e81. [89] Zaidi AH, Bakkes PJ, Lubelski J, Agustiandari H, Kuipers OP, Driessen AJ. The ABC-type multidrug resistance transporter LmrCD is responsible for an extrusion-based mechanism of bile acid resistance in Lactococcus lactis. J Bacteriol 2008;190:7357e66. [90] Agustiandari H, Lubelski J, van den Berg van Saparoea HB, Kuipers OP, Driessen AJ. LmrR is a transcriptional repressor of expression of the multidrug ABC transporter LmrCD in Lactococcus lactis. J Bacteriol 2008;190: 759e63. [91] Madoori PK, Agustiandari H, Driessen AJ, Thunnissen AM. Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition. EMBO J 2009;28:156e66. [92] Reilman E, Mars RA, van Dijl JM, Denham EL. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism. Nucleic Acids Res 2014;42:11393e407. [93] Marrer E, Satoh AT, Johnson MM, Piddock LJ, Page MG. Global transcriptome analysis of the responses of a fluoroquinolone-resistant Streptococcus pneumoniae mutant and its parent to ciprofloxacin. Antimicrob Agents Chemother 2006;50:269e78. [94] Garvey MI, Baylay AJ, Wong RL, Piddock LJ. Overexpression of patA and patB, which encode ABC transporters, is associated with fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother 2011;55:190e6. [95] Lupien A, Billal DS, Fani F, Soualhine H, Zhanel GG, Leprohon P, et al. Genomic characterization of ciprofloxacin resistance in a laboratory-derived mutant and a clinical isolate of Streptococcus pneumoniae. Antimicrob Agents Chemother 2013;57:4911e9. [96] Lupien A, Gingras H, Bergeron MG, Leprohon P, Ouellette M. Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing. J Antimicrob Chemother 2015;70:1946e59. [97] Baylay AJ, Piddock LJ. Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator. J Antimicrob Chemother 2015;70:670e9. [98] Baylay AJ, Ivens A, Piddock LJ. A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob Agents Chemother 2015;59:3098e108. [99] Boncoeur E, Durmort C, Bernay B, Ebel C, Di Guilmi AM, Croize J, et al. PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones. Biochemistry 2012;51:7755e65. [100] Orelle C, Durmort C, Mathieu K, Duchene B, Aros S, Fenaille F, et al. A multidrug ABC transporter with a taste for GTP. Sci Rep 2018;8:2309. [101] van Wonderen JH, McMahon RM, O'Mara ML, McDevitt CA, Thomson AJ, Kerr ID, et al. The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis. FEBS J 2014;281:2190e201. [102] Ward A, Reyes CL, Yu J, Roth CB, Chang G. Flexibility in the ABC transporter MsbA: alternating access with a twist. Proc Natl Acad Sci U S A 2007;104: 19005e10. [103] Zou P, McHaourab HS. Alternating access of the putative substrate-binding chamber in the ABC transporter MsbA. J Mol Biol 2009;393:574e85. [104] Choudhury HG, Tong Z, Mathavan I, Li Y, Iwata S, Zirah S, et al. Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state. Proc Natl Acad Sci U S A 2014;111:9145e50. [105] Perez C, Gerber S, Boilevin J, Bucher M, Darbre T, Aebi M, et al. Structure and mechanism of an active lipid-linked oligosaccharide flippase. Nature 2015;524:433e8. [106] Mi W, Li Y, Yoon SH, Ernst RK, Walz T, Liao M. Structural basis of MsbAmediated lipopolysaccharide transport. Nature 2017;549:233e7.

Please cite this article as: C. Orelle et al., Multidrug ABC transporters in bacteria, Research in Microbiology, https://doi.org/10.1016/ j.resmic.2019.06.001

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C. Orelle et al. / Research in Microbiology xxx (xxxx) xxx

[107] Martin C, Higgins CF, Callaghan R. The vinblastine binding site adopts highand low-affinity conformations during a transport cycle of P-glycoprotein. Biochemistry 2001;40:15733e42. [108] Ramachandra M, Ambudkar SV, Chen D, Hrycyna CA, Dey S, Gottesman MM, et al. Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state. Biochemistry 1998;37:5010e9. [109] Velamakanni S, Yao Y, Gutmann DA, van Veen HW. Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus. Biochemistry 2008;47:9300e8. [110] Hohl M, Briand C, Grutter MG, Seeger MA. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nat Struct Mol Biol 2012;19:395e402. [111] Li J, Jaimes KF, Aller SG. Refined structures of mouse P-glycoprotein. Protein Sci 2014;23:34e46. [112] Lee JY, Kinch LN, Borek DM, Wang J, Wang J, Urbatsch IL, et al. Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 2016;533: 561e4. [113] Taylor NMI, Manolaridis I, Jackson SM, Kowal J, Stahlberg H, Locher KP. Structure of the human multidrug transporter ABCG2. Nature 2017;546: 504e9. [114] Chufan EE, Sim HM, Ambudkar SV. Molecular basis of the polyspecificity of Pglycoprotein (ABCB1): recent biochemical and structural studies. Adv Cancer Res 2015;125:71e96. [115] Orelle C, Jault J-M. Structures and transport mechanisms of the ABC efflux pumps. In: Li X-Z, Elkins CA, Zgurskaya HI, editors. Efflux-mediated antimicrobial resistance in bacteria: mechanisms, regulation and clinical implications. Cham: Springer International Publishing; 2016. p. 73e98. [116] Szollosi D, Rose-Sperling D, Hellmich UA, Stockner T. Comparison of mechanistic transport cycle models of ABC exporters. Biochim Biophys Acta Biomembr 2018;1860:818e32. [117] Shapiro AB, Ling V. Positively cooperative sites for drug transport by Pglycoprotein with distinct drug specificities. Eur J Biochem 1997;250:130e7. [118] Rahman SJ, Kaur P. Conformational changes in a multidrug resistance ABC transporter DrrAB: fluorescence-based approaches to study substrate binding. Arch Biochem Biophys 2018;658:31e45. [119] Brown K, Li W, Kaur P. Role of aromatic and negatively charged residues of DrrB in multisubstrate specificity conferred by the DrrAB system of Streptomyces peucetius. Biochemistry 2017;56:1921e31. [120] Schumacher MA, Brennan RG. Structural mechanisms of multidrug recognition and regulation by bacterial multidrug transcription factors. Mol Microbiol 2002;45:885e93. [121] Schumacher MA, Miller MC, Brennan RG. Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein. EMBO J 2004;23:2923e30. [122] Redhu AK, Banerjee A, Shah AH, Moreno A, Rawal MK, Nair R, et al. Molecular basis of substrate polyspecificity of the Candida albicans Mdr1p multidrug/ H(þ) antiporter. J Mol Biol 2018;430:682e94. [123] Higgins CF, Gottesman MM. Is the multidrug transporter a flippase? Trends Biochem Sci 1992;17:18e21. [124] Gatlik-Landwojtowicz E, Aanismaa P, Seelig A. Quantification and characterization of P-glycoprotein-substrate interactions. Biochemistry 2006;45: 3020e32. [125] Hellmich UA, Lyubenova S, Kaltenborn E, Doshi R, van Veen HW, Prisner TF, et al. Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR Spectroscopy. J Am Chem Soc 2012;134(13):5857e62. [126] Li MJ, Guttman M, Atkins WM. Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale. J Biol Chem 2018;293:6297e307. [127] Mehmood S, Domene C, Forest E, Jault JM. Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations. Proc Natl Acad Sci U S A 2012;109:10832e6. [128] Wen PC, Verhalen B, Wilkens S, McHaourab HS, Tajkhorshid E. On the origin of large flexibility of P-glycoprotein in the inward-facing state. J Biol Chem 2013;288:19211e20. [129] Esser L, Zhou F, Pluchino KM, Shiloach J, Ma J, Tang WK, et al. Structures of the multidrug transporter P-glycoprotein reveal asymmetric ATP binding and the mechanism of polyspecificity. J Biol Chem 2017;292:446e61. [130] Ernst R, Kueppers P, Stindt J, Kuchler K, Schmitt L. Multidrug efflux pumps: substrate selection in ATP-binding cassette multidrug efflux pumps–first come, first served? FEBS J 2010;277:540e9. [131] Gupta RP, Kueppers P, Schmitt L, Ernst R. The multidrug transporter Pdr5: a molecular diode? Biol Chem 2011;392:53e60. [132] Galian C, Manon F, Dezi M, Torres C, Ebel C, Levy D, et al. Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. PLoS One 2011;6:e19677. [133] Doshi R, van Veen HW. Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA. J Biol Chem 2013;288:21638e47. [134] Zoghbi ME, Mok L, Swartz DJ, Singh A, Fendley GA, Urbatsch IL, et al. Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer-associated P-glycoprotein during ATP hydrolysis. J Biol Chem 2017;292:20412e24. [135] Muneyuki E, Noji H, Amano T, Masaike T, Yoshida M. F(0)F(1)-ATP synthase: general structural features of 'ATP-engine' and a problem on free energy transduction. Biochim Biophys Acta 2000;1458:467e81.

[136] Orelle C, Dalmas O, Gros P, Di Pietro A, Jault JM. The conserved glutamate residue adjacent to the walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA. J Biol Chem 2003;278: 47002e8. [137] Priess M, Goddeke H, Groenhof G, Schafer LV. Molecular mechanism of ATP hydrolysis in an ABC transporter. ACS Cent Sci 2018;4:1334e43. [138] Huang W, Liao JL. Catalytic mechanism of the maltose transporter hydrolyzing ATP. Biochemistry 2016;55:224e31. [139] Zhou Y, Ojeda-May P, Pu J. H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB. Phys Chem Chem Phys 2013;15:15811e5. [140] Schmitt L, Benabdelhak H, Blight MA, Holland IB, Stubbs MT. Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains. J Mol Biol 2003;330:333e42. [141] Seelig A. A general pattern for substrate recognition by P-glycoprotein. Eur J Biochem 1998;251:252e61. [142] Eckford PD, Sharom FJ. ABC efflux pump-based resistance to chemotherapy drugs. Chem Rev 2009;109:2989e3011. [143] Shapiro AB, Corder AB, Ling V. P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer. Eur J Biochem 1997;250:115e21. [144] Eytan GD, Regev R, Assaraf YG. Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis. J Biol Chem 1996;271:3172e8. [145] Ambudkar SV, Cardarelli CO, Pashinsky I, Stein WD. Relation between the turnover number for vinblastine transport and for vinblastine-stimulated ATP hydrolysis by human P-glycoprotein. J Biol Chem 1997;272:21160e6. [146] Herget M, Kreissig N, Kolbe C, Scholz C, Tampe R, Abele R. Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis. J Biol Chem 2009;284:33740e9. [147] Davidson AL, Shuman HA, Nikaido H. Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci U S A 1992;89:2360e4. [148] Vigonsky E, Ovcharenko E, Lewinson O. Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins. Proc Natl Acad Sci U S A 2013;110:5440e5. [149] Mimmack ML, Gallagher MP, Pearce SR, Hyde SC, Booth IR, Higgins CF. Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo. Proc Natl Acad Sci U S A 1989;86:8257e61. [150] Patzlaff JS, van der Heide T, Poolman B. The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA. J Biol Chem 2003;278:29546e51. [151] Borths EL, Poolman B, Hvorup RN, Locher KP, Rees DC. In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake. Biochemistry 2005;44:16301e9. [152] Lewinson O, Livnat-Levanon N. Mechanism of action of ABC importers: conservation, divergence, and physiological adaptations. J Mol Biol 2017;429:606e19. [153] Zoghbi ME, Altenberg GA. Luminescence resonance energy transfer spectroscopy of ATP-binding cassette proteins. Biochim Biophys Acta Biomembr 2018;1860:854e67. [154] Zoghbi ME, Fuson KL, Sutton RB, Altenberg GA. Kinetics of the association/ dissociation cycle of an ATP-binding cassette nucleotide-binding domain. J Biol Chem 2012;287:4157e64. [155] Orelle C, Alvarez FJ, Oldham ML, Orelle A, Wiley TE, Chen J, et al. Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter. Proc Natl Acad Sci U S A 2010;107:20293e8. [156] Urbatsch IL, Sankaran B, Bhagat S, Senior AE. Both P-glycoprotein nucleotidebinding sites are catalytically active. J Biol Chem 1995;270:26956e61. [157] Loo TW, Clarke DM. Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. J Biol Chem 1994;269:7750e5. [158] Sharma S, Davidson AL. Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis. J Bacteriol 2000;182:6570e6. [159] Urbatsch IL, Sankaran B, Weber J, Senior AE. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J Biol Chem 1995;270:19383e90. [160] Qu Q, Sharom FJ. Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet. Biochemistry 2002;41:4744e52. [161] Senior AE, al-Shawi MK, Urbatsch IL. The catalytic cycle of P-glycoprotein. FEBS Lett 1995;377:285e9. [162] Tombline G, Muharemagic A, White LB, Senior AE. Involvement of the “occluded nucleotide conformation” of p-glycoprotein in the catalytic pathway. Biochemistry 2005;44:12879e86. [163] Oloo EO, Tieleman DP. Conformational transitions induced by the binding of MgATP to the vitamin B12 ATP-binding cassette (ABC) transporter BtuCD. J Biol Chem 2004;279:45013e9. [164] Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, et al. A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer. Embo J 2006;25:3432e43. [165] Aittoniemi J, de Wet H, Ashcroft FM, Sansom MS. Asymmetric switching in a homodimeric ABC transporter: a simulation study. PLoS Comput Biol 2010;6: e1000762.

Please cite this article as: C. Orelle et al., Multidrug ABC transporters in bacteria, Research in Microbiology, https://doi.org/10.1016/ j.resmic.2019.06.001

C. Orelle et al. / Research in Microbiology xxx (xxxx) xxx [166] Sauna ZE, Ambudkar SV. Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human Pglycoprotein. Proc Natl Acad Sci U S A 2000;97:2515e20. [167] Higgins CF, Linton KJ. The ATP switch model for ABC transporters. Nat Struct Mol Biol 2004;11:918e26. [168] Kim Y, Chen J. Molecular structure of human P-glycoprotein in the ATPbound, outward-facing conformation. Science (New York, N.Y) 2018;359: 915e9. [169] Orelle C, Gubellini F, Durand A, Marco S, Levy D, Gros P, et al. Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA. Biochemistry 2008;47:2404e12. [170] Goddeke H, Timachi MH, Hutter CAJ, Galazzo L, Seeger MA, Karttunen M, et al. Atomistic mechanism of large-scale conformational transition in a heterodimeric ABC exporter. J Am Chem Soc 2018;140:4543e51. [171] Hutter CAJ, Timachi MH, Hurlimann LM, Zimmermann I, Egloff P, Goddeke H, et al. The extracellular gate shapes the energy profile of an ABC exporter. Nat Commun 2019;10:2260. [172] Jones PM, George AM. Molecular-dynamics simulations of the ATP/apo state of a multidrug ATP-binding cassette transporter provide a structural and

[173]

[174]

[175]

[176] [177]

11

mechanistic basis for the asymmetric occluded state. Biophys J 2011;100: 3025e34. Collauto A, Mishra S, Litvinov A, McHaourab HS, Goldfarb D. Direct spectroscopic detection of ATP turnover reveals mechanistic divergence of ABC exporters. Structure 2017;25:1264e12674 e3. Mishra S, Verhalen B, Stein RA, Wen PC, Tajkhorshid E, McHaourab HS. Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter. Elife 2014;3:e02740. Barth K, Hank S, Spindler PE, Prisner TF, Tampe R, Joseph B. Conformational coupling and trans-inhibition in the human antigen transporter ortholog TmrAB resolved with dipolar EPR spectroscopy. J Am Chem Soc 2018;140: 4527e33. Timachi MH, Hutter CA, Hohl M, Assafa T, Bohm S, Mittal A, et al. Exploring conformational equilibria of a heterodimeric ABC transporter. Elife 2017;6. Gupta RP, Kueppers P, Hanekop N, Schmitt L. Generating symmetry in the asymmetric ATP-binding cassette (ABC) transporter Pdr5 from Saccharomyces cerevisiae. J Biol Chem 2014;289:15272e9.

Please cite this article as: C. Orelle et al., Multidrug ABC transporters in bacteria, Research in Microbiology, https://doi.org/10.1016/ j.resmic.2019.06.001