Cedric Orelle
email: cedric.orelle@ibcp.fr

Khadija Mathieu

Jean-Michel Jault
email: jean-michel.jault@ibcp.fr

Cédric Orelle

Multidrug ABC transporters in bacteria

Keywords: ABC transporter, antibiotic resistance, multidrug resistance, efflux pumps

diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Multidrug efflux pumps from various families can cause antibiotic resistance in bacteria [START_REF] Chitsaz | The role played by drug efflux pumps in bacterial multidrug resistance[END_REF]. 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 [START_REF] El Meouche | Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation[END_REF][START_REF] Frimodt-Moller | Mutations causing low level antibiotic resistance ensure bacterial survival in antibiotic-treated hosts[END_REF][START_REF] Schmalstieg | The antibiotic resistance arrow of time: efflux pump induction is a general first step in the evolution of mycobacterial drug resistance[END_REF]. 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 [START_REF] Lubelski | Distribution and physiology of ABC-type transporters contributing to multidrug resistance in bacteria[END_REF]. 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 found on different combinations, being either borne on four separate polypeptides, or fused together as exemplified in the archetype of multidrug efflux pumps, the eukaryotic P-glycoprotein (Pgp, ABCB1 or MDR1). In bacteria, multidrug efflux pumps often have one TMD fused to one NBD and they can form either homodimers or heterodimers.

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-Mg or analogues 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 [START_REF] Dayan | Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein. Overexpression, purification, and role of cysteine 430[END_REF], MRP [START_REF] Ramaen | 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[END_REF], TAP1/TAP2 [START_REF] Gaudet | Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing[END_REF] or a related yeast transporter [START_REF] Janas | The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p[END_REF]) but also with NBDs from importers or from an ABC protein involved in Fe-S cluster assembly, i.e. NBDs that stand alone as full polypeptides [START_REF] Verdon | Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotidefree and nucleotide-bound conformations[END_REF][START_REF] Watanabe | Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8[END_REF][START_REF] Karpowich | Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter[END_REF]. 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 [START_REF] Hung | Crystal structure of the ATP-binding subunit of an ABC transporter[END_REF]. 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 [START_REF] Karpowich | Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter[END_REF].

The first is a RecA-like subdomain present in many NTPases [START_REF] Geourjon | A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies[END_REF][START_REF] Ye | RecA-like motor ATPases--lessons from structures[END_REF] and that carries the Walker-A (GX 4 GKT/S) and Walker-B (hy 4 D) motifs (Fig. 1A). Additional motifs are present in the RecA-like subdomain: (i) The Q-loop, a stretch of ~8 amino acids starting with a conserved glutamine and connecting the RecA-like and α-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 phosphate of ATP, the attacking water and the catalytic glutamate [START_REF] Zaitseva | H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB[END_REF]; (iii) The D-loop is located downstream of the Walker B motif. It has a central role in the functionality of ABC transporters [START_REF] Hurlimann | Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD[END_REF][START_REF] Schultz | Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA[END_REF]. When the NBDs are sufficiently close to each other, the D-loops establish a complex electrostatic and hydrogen bond network with the Walker A motif and Hloop of the opposite NBD, thereby allowing communication between the NBDs (see NBD dimerization below) [START_REF] Hohl | Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter[END_REF]. Because of its position at the dimer interface, the "D-loop" originally referred to "dimer" [START_REF] Hopfner | Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily[END_REF] 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 [START_REF] Jones | Role of the D-loops in allosteric control of ATP hydrolysis in an ABC transporter. The journal of physical chemistry[END_REF][START_REF] Oldham | Snapshots of the maltose transporter during ATP hydrolysis[END_REF]. Finally, the D-loop is also involved in the directionality and energetic of the transport [START_REF] Grossmann | Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter[END_REF].

The two other subdomains are specific to the ABC family. One is the -helical subdomain, which contains the family signature motif, also known as the C-loop. Its sequence usually starts with LSGGQ and belongs to a loop located at the N-terminus of 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 [START_REF] Dawson | Structure of a bacterial multidrug ABC transporter[END_REF]. It is only present in exporters and precedes the signature motif in the -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 [START_REF] Becker | Dynamics and structural changes induced by ATP binding in SAV1866, a bacterial ABC exporter[END_REF][START_REF] Damas | Structural consequences of ATP hydrolysis on the ABC transporter NBD dimer: molecular dynamics studies of HlyB[END_REF][START_REF] Oancea | Structural arrangement of the transmission interface in the antigen ABC transport complex TAP[END_REF][START_REF] Xu | Unidirectional Transport Mechanism in an ATP Dependent Exporter[END_REF] and this was recently demonstrated [START_REF] Lacabanne | Flexible-torigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis[END_REF].

The other subdomain is called the -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 [START_REF] Geourjon | A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies[END_REF][START_REF] Ambudkar | The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding[END_REF].

NBD dimerization is required for ATPase activity

ATP binding to isolated NBDs usually occurs with a low affinity but induces notable structural changes within the domains [START_REF] Hellmich | Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study[END_REF]. Nevertheless, the ATPase activities that could be measured, if any, were often several orders of magnitude lower than in the whole transporters [START_REF] Dayan | Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein. Overexpression, purification, and role of cysteine 430[END_REF][START_REF] Ramaen | 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[END_REF][START_REF] Watanabe | Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8[END_REF][START_REF] Procko | Distinct structural and functional properties of the ATPase sites in an asymmetric ABC transporter[END_REF]. 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 [START_REF] Moody | Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters[END_REF] (Fig. 1B-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 [START_REF] Van Der Does | Kinetics of the ATP hydrolysis cycle of the nucleotide-binding domain of Mdl1 studied by a novel site-specific labeling technique[END_REF]. 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 or MJ1267 [START_REF] Moody | Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters[END_REF] or in the MBP-MalFGK 2 transporter [START_REF] Oldham | Crystal structure of a catalytic intermediate of the maltose transporter[END_REF], or the histidine of the H-loop in the isolated NBD of HlyB [START_REF] Zaitseva | Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter[END_REF].

Both mutants retained the ability to bind ATP that triggered the dimerization of the two NBDs in an ATP-dependent process. This resulted in a NBD dimer in a head-to-tail orientation that sandwiched two ATP molecules at their interface [START_REF] Zaitseva | H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB[END_REF][START_REF] Smith | ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer[END_REF] (Fig. 1B-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-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 [START_REF] Verdon | Formation of the productive ATP-Mg2+-bound dimer of GlcV, an ABC-ATPase from Sulfolobus solfataricus[END_REF][START_REF] Buchaklian | Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA[END_REF][START_REF] Tombline | Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis[END_REF] or NMR studies [START_REF] Ramaen | 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[END_REF]. 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 [START_REF] Chen | A tweezers-like motion of the ATPbinding cassette dimer in an ABC transport cycle[END_REF]. 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 [START_REF] Chen | A tweezers-like motion of the ATPbinding cassette dimer in an ABC transport cycle[END_REF]. In addition, it was observed that in the presence of ATP and orthovanadate, leading to an inhibited state mimicking the post-hydrolyis step (i.e. an ADP-Mg-Vi trapped conformation), UV irradiation induced the photocleavage in both 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 [START_REF] Fetsch | Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter[END_REF].

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 [START_REF] Oldham | Snapshots of the maltose transporter during ATP hydrolysis[END_REF][START_REF] Bukowska | A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter[END_REF].

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 [START_REF] Hassan | Active export proteins mediating drug resistance in staphylococci[END_REF][START_REF] Paulsen | Comparative genomics of microbial drug efflux systems[END_REF]. 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 [START_REF] Gebhard | ABC transporters of antimicrobial peptides in Firmicutes bacteriaphylogeny, function and regulation[END_REF]). 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.

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 [START_REF] Van Veen | Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1[END_REF]. LmrA functions as a homodimer [START_REF] Van Veen | The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating twosite (two-cylinder engine) mechanism[END_REF]. Each monomer is made of one transmembrane domain containing 6 predicted transmembrane helices and one nucleotide-binding 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 and TPP + [START_REF] Van Veen | Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1[END_REF]. 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 [START_REF] Agboh | Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force[END_REF].

Based on its homology with LmrA, an uncharacterized membrane protein from Bacillus subtilis was investigated [START_REF] Steinfels | Highly efficient over-production in E. coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis[END_REF]. Its functional overexpression in E. coli membranes [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF] 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 [START_REF] Dalmas | Time-resolved fluorescence resonance energy transfer shows that the bacterial multidrug ABC halftransporter BmrA functions as a homodimer[END_REF] was renamed BmrA (Bacillus multidrug resistance ATP) [START_REF] Steinfels | Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis[END_REF]. Interestingly, a Bacillus subtilis strain resistant to the antibiotic cervimycin C was isolated, in which a promoter mutation upregulated the expression of bmrA [START_REF] Krugel | Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA[END_REF].

MacAB/TolC from E. coli was initially characterized as a macrolide-specific tripartite pump [START_REF] Kobayashi | Novel macrolide-specific ABC-type efflux transporter in Escherichia coli[END_REF]. TolC is an outer membrane porin, while MacA is a Membrane Fusion Protein that interacts with both partner proteins [START_REF] Fitzpatrick | Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump[END_REF]. Homologues are present in various Gram-negative bacteria [START_REF] Okada | Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii[END_REF][START_REF] Rouquette-Loughlin | Characterization of the MacA-MacB efflux system in Neisseria gonorrhoeae[END_REF]. MacB has an inverted topology: a N-terminal NBD is followed by a Cterminal TMD containing 4 transmembrane helices [START_REF] Crow | Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily[END_REF][START_REF] Okada | Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii[END_REF]. Nanomolar affinity interactions occur between TolC and MacA, and between MacA and MacB [START_REF] Lu | Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter[END_REF]. MacB is a dimer [START_REF] Lin | MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA[END_REF] whose ATPase activity is strongly stimulated by MacA [START_REF] Tikhonova | Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB[END_REF]. Several drug-unrelated physiological functions have been proposed, including protoporphyrin [START_REF] Turlin | Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli[END_REF] and enterotoxin STII export [START_REF] Crow | Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily[END_REF][START_REF] Yamanaka | MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II[END_REF]. 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 [START_REF] Crow | Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily[END_REF]. 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 also proposed that other ABC proteins might also use mechanotransmission to function [START_REF] Greene | Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective[END_REF].

DrrAB from Streptomyces peucetius exports daunorubicin and doxorubicin, two antibiotics used as anticancer agents, that this bacterium produces [START_REF] Guilfoile | A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin[END_REF]. 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 [START_REF] Li | The DrrAB efflux system of Streptomyces peucetius is a multidrug transporter of broad substrate specificity[END_REF]. 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 [START_REF] Choudhuri | Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis[END_REF]. In contrast to LmrA and BmrA, 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 [START_REF] Gandlur | Membrane topology of the DrrB protein of the doxorubicin transporter of Streptomyces peucetius[END_REF]. Cross-linking experiments suggested a stoichiometry of 2 TMDs and 2 NBDs [START_REF] Kaur | Biochemical coupling between the DrrA and DrrB proteins of the doxorubicin efflux pump of Streptomyces peucetius[END_REF].

Transporters with asymmetric NBDs

Many multidrug ABC transporters have a degenerate ATP-binding site [START_REF] Lubelski | Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric[END_REF][START_REF] Zutz | Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus[END_REF] 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 non-consensual residues (Fig. 1B-C). Consequently, the functioning mechanism of these heterodimers is asymmetric with the degenerate NDB being poorly active in ATP hydrolysis [START_REF] Hurlimann | Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD[END_REF][START_REF] Lubelski | Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric[END_REF].

LmrCD was shown to be a major multidrug resistance transporter in Lactococcus lactis [START_REF] Lubelski | ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassette-type multidrug transporter[END_REF][START_REF] Lubelski | LmrCD is a major multidrug resistance transporter in Lactococcus lactis[END_REF][START_REF] Zaidi | The ABCtype multidrug resistance transporter LmrCD is responsible for an extrusion-based mechanism of bile acid resistance in Lactococcus lactis[END_REF] and its expression is under the control of a transcriptional repressor named LmrR [START_REF] Agustiandari | LmrR is a transcriptional repressor of expression of the multidrug ABC transporter LmrCD in Lactococcus lactis[END_REF]. Binding of drugs to LmrR reduces its affinity for LmrCD promoter thereby inducing the expression of the transporter [START_REF] Madoori | Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition[END_REF].

BmrCD is a Bacillus subtilis transporter [START_REF] Torres | The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter[END_REF], and antibiotics targeting protein synthesis induce its expression through a ribosome-mediated transcriptional attenuation mechanism [START_REF] Reilman | The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism[END_REF]. When overexpressed in E. coli membranes, BmrCD transports several drugs such as Hoechst 33342, doxorubicin or mitoxantrone [START_REF] Torres | The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter[END_REF].

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 [START_REF] Robertson | Use of an efflux-deficient streptococcus pneumoniae strain panel to identify ABC-class multidrug transporters involved in intrinsic resistance to antimicrobial agents[END_REF]. After exposing a laboratory strain to ciprofloxacin, a multidrug resistant strain was isolated and found to upregulate the expression of PatA and PatB genes [START_REF] Marrer | Global transcriptome analysis of the responses of a fluoroquinolone-resistant Streptococcus pneumoniae mutant and its parent to ciprofloxacin[END_REF]. Later, such upregulation was also found in clinical isolates resistant to fluoroquinolones [START_REF] Garvey | Overexpression of patA and patB, which encode ABC transporters, is associated with fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae[END_REF][START_REF] Lupien | Genomic characterization of ciprofloxacin resistance in a laboratory-derived mutant and a clinical isolate of Streptococcus pneumoniae[END_REF]. Several mechanisms were described for PatAB upregulation: promoter region and internal mutations [START_REF] Lupien | Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing[END_REF], disruption of a transcriptional attenuator [START_REF] Baylay | Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator[END_REF] or gene duplication [START_REF] Baylay | A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae[END_REF]. Studies of the transporter overexpressed in E. coli showed that only the heterodimer was functional for drug efflux [START_REF] Boncoeur | PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones[END_REF] and that PatAB strongly favors GTP as energy source to efflux drugs, especially at 37 °C [START_REF] Orelle | A multidrug ABC transporter with a taste for GTP[END_REF]. Interestingly, intracellular concentrations of ATP and GTP in Streptococcus pneumonia were both in mM ranges, and fluoroquinolone treatment substantially increased GTP concentrations [START_REF] Orelle | A multidrug ABC transporter with a taste for GTP[END_REF].

EfrCD is a MDR transporter recently identified in Enterococcus faecalis [START_REF] Hurlimann | The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis[END_REF]. When the genes encoding this transporter were inactivated, the mutated strain displayed increased sensitivity to several drugs, including Hoechst 3342, daunorubicin, doxorubicin and ethidium.

Similar results were obtained when the transporter was heterogeneously overexpressed in a Lactococcus lactis strain hypersensitive to drugs due to the disruption of LmrA and LmrCD genes.

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 [START_REF] Van Wonderen | The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis[END_REF][START_REF] Ward | Flexibility in the ABC transporter MsbA: Alternating access with a twist[END_REF][START_REF] Zou | Alternating access of the putative substrate-binding chamber in the ABC transporter MsbA[END_REF]. Of note, a third occluded conformational state was also observed for McjD [START_REF] Choudhury | Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state[END_REF], PglK [START_REF] Perez | Structure and mechanism of an active lipid-linked oligosaccharide flippase[END_REF] and MsbA [START_REF] Mi | Structural basis of MsbA-mediated lipopolysaccharide transport[END_REF] 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 [START_REF] Van Veen | The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating twosite (two-cylinder engine) mechanism[END_REF][START_REF] Martin | The vinblastine binding site adopts high-and lowaffinity conformations during a transport cycle of P-glycoprotein[END_REF][START_REF] Ramachandra | Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state[END_REF].

Type I exporters. Sav1866 [START_REF] Dawson | Structure of a bacterial multidrug ABC transporter[END_REF][START_REF] Velamakanni | Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus[END_REF] and TM287/288 [START_REF] Hohl | Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation[END_REF] 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 [START_REF] Li | Refined structures of mouse P-glycoprotein[END_REF], 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 inwardfacing state, a central cavity is accessible from the cytoplasm. In the outward-facing 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 ( 25Å). 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 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 lipid-linked oligosaccharide flippase PglK [START_REF] Perez | Structure and mechanism of an active lipid-linked oligosaccharide flippase[END_REF].

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 [START_REF] Lee | Crystal structure of the human sterol transporter ABCG5/ABCG8[END_REF][START_REF] Taylor | Structure of the human multidrug transporter ABCG2[END_REF]. 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 [START_REF] Greene | Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective[END_REF].

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 [START_REF] Chufan | Molecular Basis of the Polyspecificity of P-Glycoprotein (ABCB1): Recent Biochemical and Structural Studies[END_REF][START_REF] Orelle | Structures and Transport Mechanisms of the ABC Efflux Pumps[END_REF][START_REF] Szollosi | Comparison of mechanistic transport cycle models of ABC exporters[END_REF]).

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 [START_REF] Shapiro | Positively cooperative sites for drug transport by P-glycoprotein with distinct drug specificities[END_REF]. This property is unique to MDR transporters and was also found for bacterial transporters such as LmrA [START_REF] Van Veen | The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating twosite (two-cylinder engine) mechanism[END_REF] or BmrA [START_REF] Steinfels | Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis[END_REF]. Drug binding studies also supported that DrrAB transporter contains multiple drug binding sites [START_REF] Rahman | Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding[END_REF]. 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 [START_REF] Brown | Role of Aromatic and Negatively Charged Residues of DrrB in Multisubstrate Specificity Conferred by the DrrAB System of Streptomyces peucetius[END_REF]. Interestingly, some transcriptional repressors of drug-efflux pumps also display this polyspecificity [START_REF] Madoori | Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition[END_REF]. Their co-crystallization with different drugs illustrated the plasticity of a large multidrug-binding pocket that can accommodate several drugs, in some cases simultaneously [START_REF] Schumacher | Structural mechanisms of multidrug recognition and regulation by bacterial multidrug transcription factors[END_REF][START_REF] Schumacher | Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein[END_REF]. 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 [START_REF] Redhu | Molecular Basis of Substrate Polyspecificity of the Candida albicans Mdr1p Multidrug/H(+) Antiporter[END_REF].

The second explanation derives from the vacuum cleaner model that has been proposed for P-gp [START_REF] Higgins | Is the multidrug transporter a flippase?[END_REF]. Due to their hydrophobicity and high membrane partition coefficient, drugs accumulate in the membrane up to several orders of magnitudes [START_REF] Gatlik-Landwojtowicz | Quantification and characterization of P-glycoprotein-substrate interactions[END_REF].

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 [START_REF] Hellmich | Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR Spectroscopy[END_REF][START_REF] Li | Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale[END_REF][START_REF] Mehmood | Dynamics of a bacterial multidrug ABC transporter in the inward-and outward-facing conformations[END_REF][START_REF] Wen | On the origin of large flexibility of P-glycoprotein in the inward-facing state[END_REF]. Conformational selection may provide a source of substrate promiscuity and the constant remodeling of the drug-binding 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 [START_REF] Esser | Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity[END_REF].

Finally, Ernst and collaborators have proposed a kinetic substrate selection model [START_REF] Ernst | Multidrug efflux pumps: substrate selection in ATP-binding cassette multidrug efflux pumps--first come, first served?[END_REF][START_REF] Gupta | The multidrug transporter Pdr5: a molecular diode?[END_REF]. 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 k on and k off , 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 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 [START_REF] Hurlimann | The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis[END_REF][START_REF] Lubelski | LmrCD is a major multidrug resistance transporter in Lactococcus lactis[END_REF] 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 [START_REF] Robertson | Use of an efflux-deficient streptococcus pneumoniae strain panel to identify ABC-class multidrug transporters involved in intrinsic resistance to antimicrobial agents[END_REF]. This may be one of the reasons explaining the high number of drug transporters with various specificities in bacterial genomes.

ATPase activity and transport mechanism

Multidrug transporters typically display a high basal ATPase activity, which for bacterial transporters is often moderately stimulated by drugs [START_REF] Steinfels | Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis[END_REF][START_REF] Hurlimann | The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis[END_REF][START_REF] Galian | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization[END_REF]. However, several lines of evidence suggest that drugs binding facilitate the dimerization of the nucleotidebinding domains thereby stimulating ATP hydrolysis [START_REF] Doshi | Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA[END_REF][START_REF] Zoghbi | Substrateinduced conformational changes in the nucleotide-binding domains of lipid bilayer-associated P-glycoprotein during ATP hydrolysis[END_REF].

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 [START_REF] Zaitseva | H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB[END_REF]. 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 [START_REF] Geourjon | A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies[END_REF][START_REF] Muneyuki | F(0)F(1)-ATP synthase: general structural features of 'ATP-engine' and a problem on free energy transduction[END_REF]. 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 [START_REF] Verdon | Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotidefree and nucleotide-bound conformations[END_REF][START_REF] Hung | Crystal structure of the ATP-binding subunit of an ABC transporter[END_REF][START_REF] Oldham | Snapshots of the maltose transporter during ATP hydrolysis[END_REF]. Mutagenesis and kinetics studies substantiated the direct role of this Glu residue in the ATPase activity of ABC transporters [START_REF] Moody | Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters[END_REF], and its replacement by several other residues in BmrA (e.g. Asp, Gln, Ala…) invariably led to a transporter devoid of ATPase activity [START_REF] Orelle | The Conserved Glutamate Residue Adjacent to the Walker-B Motif Is the Catalytic Base for ATP Hydrolysis in the ATP-binding Cassette Transporter BmrA[END_REF]. 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 [START_REF] Priess | Molecular Mechanism of ATP Hydrolysis in an ABC Transporter[END_REF]. 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 [START_REF] Huang | Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP[END_REF], 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 [START_REF] Zaitseva | H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB[END_REF]. This, together with the lack of effect of D 2 O on the ATPase activity of the HlyB NBD [START_REF] Zaitseva | H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB[END_REF], lent support to a substrate-assisted catalysis, as also pointed out by molecular dynamics simulation on the same NBD [START_REF] Zhou | H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB[END_REF]. 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 [START_REF] Oldham | Snapshots of the maltose transporter during ATP hydrolysis[END_REF]. Possibly related to this, the conserved Glu residue of the SufC NBD is located in a very unusual 3 10 helix; this peculiar conformation flips its side-chain out of the ATPbinding site and this domain displays an extremely low basal ATPase activity (k cat  1.2 min - 1 ) [START_REF] Watanabe | Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8[END_REF]. 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 [START_REF] Schmitt | Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains[END_REF]. 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 [START_REF] Zhou | H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB[END_REF].

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 [START_REF] Seelig | A general pattern for substrate recognition by P-glycoprotein[END_REF][START_REF] Eckford | ABC efflux pump-based resistance to chemotherapy drugs[END_REF]. These molecules spontaneously partition into lipid bilayers where they bind to multidrug transporters and are effluxed in an ATP-dependent manner [START_REF] Shapiro | P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer[END_REF]. 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 [START_REF] Steinfels | Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis[END_REF][START_REF] Hurlimann | The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis[END_REF][START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF][START_REF] Orelle | A multidrug ABC transporter with a taste for GTP[END_REF][START_REF] Galian | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization[END_REF]. 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. [START_REF] Steinfels | Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis[END_REF]). 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 [START_REF] Shapiro | P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer[END_REF][START_REF] Eytan | Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis[END_REF][START_REF] Ambudkar | Relation between the turnover number for vinblastine transport and for vinblastine-stimulated ATP hydrolysis by human Pglycoprotein[END_REF]. 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 [START_REF] Herget | Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis[END_REF] or type I ABC importers [START_REF] Davidson | Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins[END_REF][START_REF] Vigonsky | Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins[END_REF], their ATPase activity is strictly coupled to allocrite transport. For type I importers like MalFGK 2 or OpuA, it seems that the ATP/allocrite ratio is close to 2 [START_REF] Mimmack | Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo[END_REF][START_REF] Patzlaff | The ATP/substrate stoichiometry of the ATPbinding cassette (ABC) transporter OpuA[END_REF]. 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 [START_REF] Borths | In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake[END_REF]. 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 [START_REF] Lewinson | Mechanism of Action of ABC Importers: Conservation, Divergence, and Physiological Adaptations[END_REF]. 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 [START_REF] Zoghbi | Luminescence resonance energy transfer spectroscopy of ATP-binding cassette proteins[END_REF]. Because the two ATP-binding sites are localized at the interface of the two monomers, ATP binding promotes the formation of a closed conformation [START_REF] Moody | Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters[END_REF][START_REF] Zoghbi | Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain[END_REF]. 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 -helical subdomains within each NBD rotate toward each other upon ATP binding and move outward in the post-hydrolysis stage [START_REF] Karpowich | Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter[END_REF][START_REF] Orelle | Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter[END_REF]. Hence, the energy of ATP binding and hydrolysis is coupled to conformational changes in the TMD thereby mediating alternating access of the substratebinding site to each side of the membrane. Several mechanistic models of drug transport have been proposed [START_REF] Szollosi | Comparison of mechanistic transport cycle models of ABC exporters[END_REF]. 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 either site [START_REF] Urbatsch | Both P-glycoprotein nucleotide-binding sites are catalytically active[END_REF]. In line with this, isolated membranes containing each half of human P-gp expressed separately showed similar levels of ATPase activity [START_REF] Loo | Reconstitution of drug-stimulated ATPase activity following coexpression of each half of human P-glycoprotein as separate polypeptides[END_REF]. 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 [START_REF] Orelle | The Conserved Glutamate Residue Adjacent to the Walker-B Motif Is the Catalytic Base for ATP Hydrolysis in the ATP-binding Cassette Transporter BmrA[END_REF][START_REF] Sharma | Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis[END_REF], and this was sufficient to prevent ATP hydrolysis by P-gp [START_REF] Urbatsch | P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site[END_REF][START_REF] Qu | Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet[END_REF]. 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 [START_REF] Senior | The catalytic cycle of P-glycoprotein[END_REF]. 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 [START_REF] Tombline | Involvement of the "occluded nucleotide conformation" of p-glycoprotein in the catalytic pathway[END_REF]. The presence of one ATP molecule in a more occluded conformation was supported by molecular dynamic studies performed on BtuCD [START_REF] Oloo | Conformational transitions induced by the binding of MgATP to the vitamin B12 ATP-binding cassette (ABC) transporter BtuCD[END_REF] and also by the structure of the NBD of HlyB. This shows an asymmetry of the two ATP-binding sites in the presence of Mg 2+ and in particular the opening of a phosphate exit tunnel in one of the two sites [START_REF] Zaitseva | A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer[END_REF]. 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 [START_REF] Xu | Unidirectional Transport Mechanism in an ATP Dependent Exporter[END_REF][START_REF] Aittoniemi | Asymmetric switching in a homodimeric ABC transporter: a simulation study[END_REF]. 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 [START_REF] Sauna | Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein[END_REF]. 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 [START_REF] Higgins | The ATP switch model for ABC transporters[END_REF]. 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 [START_REF] Janas | The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p[END_REF].

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 [START_REF] Kim | Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation[END_REF], lending some support to these two models. The drug-binding cavity observed in previous inward-facing 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 [START_REF] Orelle | Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA[END_REF]. 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 [START_REF] Lacabanne | Flexible-torigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis[END_REF]. 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 outwardclosed 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 [START_REF] Xu | Unidirectional Transport Mechanism in an ATP Dependent Exporter[END_REF]. 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 [START_REF] Goddeke | Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter[END_REF]. The recent publication of TM287/TM288 structure in the outward-facing 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 [START_REF] Hutter | The extracellular gate shapes the energy profile of an ABC exporter[END_REF]. In addition, based on mutagenesis and the use of state-specific nanobodies, the authors 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 inward-facing 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 [START_REF] Jones | Molecular-dynamics simulations of the ATP/apo state of a multidrug ATP-binding cassette transporter provide a structural and mechanistic basis for the asymmetric occluded state[END_REF].

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 [START_REF] Hurlimann | Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD[END_REF][START_REF] Lubelski | Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric[END_REF][START_REF] Zutz | Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus[END_REF]. 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 [START_REF] Collauto | Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters[END_REF]. 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 [START_REF] Mishra | Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter[END_REF] unlike what is observed for TmrAB [START_REF] Barth | Conformational Coupling and trans-Inhibition in the Human Antigen Transporter Ortholog TmrAB Resolved 33[END_REF] or TM287/TM288 [START_REF] Timachi | Exploring conformational equilibria of a heterodimeric ABC transporter[END_REF], 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-Mg 2+ 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 Dloops [START_REF] Hohl | Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter[END_REF]. 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 [START_REF] Timachi | Exploring conformational equilibria of a heterodimeric ABC transporter[END_REF].

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 [START_REF] Gupta | Generating symmetry in the asymmetric ATP-binding cassette (ABC) transporter Pdr5 from Saccharomyces cerevisiae[END_REF]. 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 [START_REF] Hurlimann | Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD[END_REF]. 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 NBD-NBD or NBD-TMD communication and highlight the importance of the degenerate site in the function of the consensus one.

Vibrio cholerae VcaM

Tetracycline, norfloxacin/ciprofloxacin, doxorubicin/daunorubicine, DAPI, Hoechst 33342 [START_REF] Huda | Molecular cloning and characterization of an ABC multidrug efflux pump, VcaM, in Non-O1 Vibrio cholerae[END_REF]

Figure 1 .

 1 Figure 1. Conserved motifs found in the NBDs and the organization of the transient

Figure 2 .

 2 Figure 2. 3D structures of selected exporters from the ABC family. The N-terminal half of

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

 1

	Bacterium	Protein	Drugs transported	Selected references
			Ethidium, Hoechst 33342,
		BmrA	doxorubicin, 7-amino-actinomycin D,	[47, 48]
	Bacillus subtilis		cervimycin C
		BmrCD	Hoechst 33342, BCECF, mitoxantrone, doxorubicin	[49]
			Acriflavine, ethidium, DAPI, TPP,
		EfrAB	norfloxacin/ciprofloxacin,	[50]
	Enterococcus faecalis		doxycycline
		EfrCD	Daunorubicin, doxorubicin, ethidium, Hoechst 33342, BCECF-AM	[51]
	Escherichia coli	MacB	Macrolides, bacitracin, colistin, enterotoxin STII, protoporphyrin	[52-55]
	Lactobacillus brevis	HorA	Hop compounds, ethidium, Hoechst 33342	[56]
			Ethidium, daunomycin, rhodamine
			6G, TPP+, macrolides,
		LmrA	streptogramins,	[57, 58]
	Lactococcus lactis		tetracyclines, chloramphenicol
		LmrCD	Daunomycin, Hoechst 33342, BCECF-AM, ethidium	[59]
			Ampicillin, chloramphenicol,
		Rv0194	streptomycin, tetracycline, vancomycin, erythromycin,	[60]
	Mycobacterium tuberculosis	Rv1218c	novobiocin, ethidium Some pyridones, biaryl-piperazines, bisanilino-	[61]
			pyrimidines, pyrroles
		DrrAB	Doxorubicin, ethidium bromide, BCECF	[62]
	Serratia marcescens SmdAB	norfloxacin, tetracycline, DAPI, Hoechst 33342	[63]
	Staphylococcus aureus	AbcA	Beta-lactams, TPP, moenomycin, Phenol-soluble modulins	[64-66]
	Streptococcus mutants	VltAB	Quaternary ammonium ethidium bromide, safranin) compounds (violagen, acriflavin,	[67]
	Streptococcus pneumoniae	PatAB	Berberine, acriflavin, ethidium, norfloxacin, Hoechst 33342	[68]
	Streptococcus peucetius	DrrAB	Daunorubicin, doxorubicin, Hoechst 33342, ethidium	[69]

Acknowledgements

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

Conflict of interest

The authors declare no conflict of interest.

Figure 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.