Deciphering the molecular basis of multidrug recognition: Crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR

Research in Microbiology 154 (2003) 69–77 www.elsevier.com/locate/resmic

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Deciphering the molecular basis of multidrug recognition: Crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR Maria A. Schumacher, Richard G. Brennan ∗ Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239-3098, USA Received 11 November 2002; accepted 21 November 2002 First published online 22 November 2002

Abstract Multidrug transporters and their transcriptional regulators are key components of bacterial multidrug resistance (MDR). How these multidrug binding proteins can recognize such chemically disparate compounds represents a fascinating question from a structural standpoint and an important question in future drug development efforts. The Staphylococcus aureus multidrug binding regulator, QacR, is soluble and recognizes an especially wide range of structurally dissimilar compounds, properties making it an ideal model system for deciphering the molecular basis of multidrug recognition. Recent structures of QacR have afforded the first view of any MDR protein bound to multiple drugs, revealing key structural features of multidrug recognition, including a multisite binding pocket.  2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Multidrug recognition; QacR; Multisite model; Transcription regulation; Repressor

1. Introduction Bacterial multidrug resistance (MDR) is defined as the ability of bacteria to survive potentially lethal doses of structurally diverse drugs, which eradicate nonresistant cells. One mechanism that contributes to MDR is the active efflux of drugs by membrane-bound multidrug transporters [26,27]. In bacteria, a critical means of regulation of these transporter genes is the utilization of transcriptional activators and/or repressors [9]. Importantly, these regulators often bind many of the same structurally and chemically diverse drugs that are substrates of the pumps, which they regulate and thus, these proteins are themselves multidrug binding proteins [1,8,22]. As such, these regulators act as cytosolic multidrug sensors, which ultimately cause stimulation of the production of MDR transporters in the presence of toxic compounds. One such protein, the regulator QacR from Staphylococcus aureus, represses the transcription of the qacA multidrug pump gene by binding the IR1 operator site. QacR is induced from this site by binding one of a chemically disparate group of * Corresponding author.

E-mail address: [email protected] (R.G. Brennan).

cationic lipophilic compounds [8]. This leads to the production of more of the QacA transporter, facilitating the removal of these cytosolic toxins. Recent structures of QacR bound to the IR1 operator site and six different “drugs” have revealed not only the mechanism of induction but the molecular basis for multidrug recognition by QacR [28,29]. Moreover, these structures provide an explanation for data, which has accumulated over a decade, that indicates multidrug binding must somehow involve the utilization of multiple sites within a single protein. In this review we describe these structures and detail the fascinating lessons that they divulge about the mechanism of multidrug recognition.

2. QacR, a transcription repressor of the qacA multidrug transporter gene The S. aureus protein, QacR, is part of the qac locus, which consists of qacA (or qacB) and the divergently transcribed qacR genes. This locus is distinctive among MDR genes in that it is plasmid encoded [8]. The fact that MDR plasmids may be important in drug resistance in S. au-

0923-2508/03/$ – see front matter  2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0923-2508(02)00013-X

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reus is suggested by the recent discovery that a large number of S. aureus strains, which are unresponsive to antibiotics, also contain these DNA elements. Interestingly, the qac locus confers resistance to monovalent and bivalent cationic lipophilic antiseptics and disinfectants, which includes quaternary ammonium compounds (thus the name, Qac) [16]. In the absence of any inducing drug, the 188 residue QacR protein represses transcription of the qacA gene [5] by binding the ∼28 bp pseudo palindromic IR1 sequence, which is located downstream from the qacA promoter and overlaps its transcription start site [8]. The position of this repression complex relative to the transcription start site suggests that QacR functions by hindering the transition of the RNA polymerase-promoter complex into a processively transcribing state rather than blocking the binding of RNA polymerase. QacR is a member of the TetR family of transcriptional repressors [4]. Prior to the structure determination of QacR, TetR was the only member of this family for which structures were available [12,21]. TetR structures revealed that it contains a helix-turn-helix (HTH) DNA binding motif within an N-terminal three-helix bundle and a larger C-terminal helical domain, which binds its inducer, tetracycline. TetR members are induced by a variety of compounds and thus, it is not surprising that their C-terminal domains display little sequence homology. In contrast, the N-terminal DNA-binding domains of these proteins (∼50 residues) show strong sequence homology, and thus it would be predicted that all TetR members contain a HTH motif and bind DNA similarly to TetR [21]. Unexpectedly QacR binds an operator nearly twice the size of the “normal” 15 bp sites bound by other characterized TetR proteins [8]. Moreover, QacR is a multidrug binding protein, which is induced from its IR1 site by its interaction with a number of structurally dissimilar cationic lipophilic antiseptics, disinfectants and cytotoxins including rhodamine 6G (R6G), crystal violet (CV) and ethidium (Et). These compounds are also substrates for the QacA pump [8]. The affinity of QacR for these compounds range from 0.1 µM to over 10.0 µM [28]. Thus, initial structural studies on QacR addressed two key mechanistic features regarding its function, (1) how this regulator specifically binds its extended operator site and (2) how this protein is able to recognize such a diverse array of structurally dissimilar inducers.

scattering, suggested that two QacR dimers bind the IR1 site and that binding is likely cooperative [10]. In sum, these data indicated that QacR binds its cognate DNA site in a manner that is quite distinct from that of family member, TetR [21]. The structural mechanism that QacR utilizes to bind the 28 bp IR1 was revealed recently by the 2.90 Å resolution crystal structure of a QacR–IR1 complex [29]. The structure showed that QacR is comprised of nine helices: α1 (3–18), α2 (25–32), α3 (36–42), α4 (46–71), α5 (75–88), α6 (96–108), α7 (110–136), α8 (145–162) and α9 (168–185). Similar to TetR, the first three helices of QacR form a three-helix bundle DNA binding domain and contain a helix-turn-helix (HTH) motif (α2 and α3) (Fig. 1, top). However, as anticipated the structure revealed that the DNA binding mode of QacR is quite distinct from TetR and

2.1. QacR binds IR1 as a dimer of dimers The 28 bp IR1 site that QacR binds consists of an inverted repeat with two ∼11 bp half sites, which are separated by a 6 bp spacer [8]. Binding was found to be dependent on the sequence of the half sites as well as the correct spacing (6 bp) of the half sites [10]. However, the sequence of the 6 bp spacer is less essential for high affinity QacR binding as the spacer nucleotides can be mutated with little effect on QacR binding [10]. Surprisingly, biochemical studies, including gel filtration, protein–DNA cross-linking and dynamic light

Fig. 1. Structures of the QacR–DNA and QacR–R6G complexes. Top: the QacR–DNA complex shows the dimer of dimers DNA binding mode. The helices of one “proximal” and one “distal” subunit are labeled. Bottom: the QacR–R6G structure, which is a representative of all QacR–drug complexes that are discussed. In the QacR–R6G structure, the arrow indicates the direction in which the addition of the extra turn of helix to α5 (dark blue) drives a movement of α6 and the adjacent DNA-binding domain to cause dissociation of QacR from the IR1 site, i.e., induction. R6G is shown as sticks with nitrogen atoms colored blue, oxygens red and carbons, yellow. This figure and Figs. 3 and 4 were made using Swiss-PdbViewer [11] and rendered with POVRAY [23].

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involves two QacR dimers in the recognition of its cognate DNA site. Interestingly, despite their different DNA binding arrangements and recognition modes, QacR and TetR have similar three-dimensional structures. Indeed, the three-helix bundle DNA-binding domain of QacR can be superimposed onto that of TetR with a root mean squared deviation (r.m.s.d.) of 1.6 Å for 46 corresponding Cα atoms. Quite unexpectedly was the finding that even the inducer binding domains of these proteins contain a short region of structural similarity, which is located within two C-terminal helices that form a four helix dimerization motif; helices α8 and α9 (with α8 and α9 ) in QacR and α8 and α10 (α8 and α10 ) in TetR. These four helix regions from QacR and TetR superimpose with an r.m.s.d. of 1.4 Å despite a sequence identity of only 11%. This unexpected similarity suggests that other TetR members, in addition to containing threehelix bundle DNA-binding domains, may also dimerize via a similar four-helix motif. Additional structures of TetR family members are needed to address this possibility. 2.2. QacR–IR1 contacts The QacR–DNA structure reveals how two QacR dimers are used in IR1 recognition: each dimer docks on either side of the DNA site such that two HTH motifs, one from each dimer, contact successive major grooves, but are offset [29]. This binding mode allows extended binding and is consistent with the footprint that is observed for QacR binding [8]. QacR contacts only the DNA major groove with a total of 16 base and 44 phosphate interactions. Each QacR subunit within a given dimer docks differently onto the DNA, one distal to the dyad that relates dimers and the other more proximal to this dyad. This binding arrangement, wherein the distal subunits (magenta in Fig. 1) provide most of the base specific contacts while the proximal subunits (cyan in Fig. 1, top) make a limited number of base specific interactions outside the 6 bp spacer, is consistent with data that demonstrated the sequence of the outer inverted repeats is essential for specific binding while the central six base pairs provide the proper spacing for these repeats [8,10]. Specific recognition is effected by key base contacts made by the distal monomers that include two hydrophobic contacts and two hydrogen bonds. A notable part of the recognition process is imparted by the hydrogen bond between the amide nitrogen of Gly37 from the recognition helix to the O6 of a guanine base (G7 ). The presence of a glycine residue at this position is absolutely essential for tight docking of the recognition helix, which leaves no space for intervening solvent. Such tight packing and the attendant release of solvent, suggests a favorable entropic contribution to the energetics of complex formation, a contention supported by isothermal titration calorimetry studies on the QacR–IR1 interaction [29]. Interestingly, nucleotide G7 is the qacA transcription start site [8] and its interaction with Gly37 may be an important element of repression. In the proximal subunits, the corresponding Gly37 also makes specifying con-

Fig. 2. Electrostatic surface potential of the DNA-bound QacR. The orientation is the same view as Fig. 1. The red and blue regions indicate negative and positive charged regions, respectively. Note the highly basic nature of the DNA binding domains and the very electronegative nature of the multidrug binding pockets. The DNA is shown as sticks where nitrogen atoms are blue, oxygen red and carbon white and phosphorus yellow. This figure was made with GRASP [20].

tacts to another guanine, G11 , thus explaining a preference for a guanine at position 11. The similarity of base recognition by the distal subunit of the QacR dimer and the proximal subunit of the other QacR dimer in base preferences, which derives from contacts mediated by the corresponding Gly37 residues as well as hydrophobic-base interactions, revealed a pseudo direct repeat in the DNA bound by the HTH motifs. The last feature of DNA recognition by QacR is the high degree of structural complementarity between the surfaces of the binding partners, which is further enhanced by the complementary electrostatic fields of the highly basic DNA binding domain of QacR and the DNA phosphate backbone (Fig. 2). 2.3. Structural basis for the cooperative binding of IR1 by QacR The accumulated biochemical data on QacR–IR1 binding provide strong evidence that the QacR dimer binds the IR1 site cooperatively [10]. The QacR–DNA structure demonstrates that cooperative binding cannot be mediated by protein–protein interactions, as the closest approach of each dimer is greater than 5.0 Å. Rather, the structure indicates that cooperativity arises from the induced fit of the DNA conformation to the QacR dimer conformation. Specifically, although the QacR bound IR1 site displays B-DNAlike characteristics and a global bend of only 3.0◦ [25], the major groove is markedly widened throughout the entire binding site (average major groove width: 12.6 Å vs.

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11.0 Å for B-DNA). This major groove expansion requires a significant unwinding of the DNA, which is reflected in the observed twist of 32.1◦ and 11.2 base pairs per turn as compared to a twist of 34.3◦ and 10.5 base pairs per turn observed in canonical B-DNA. The importance of these features in the binding of IR1 by QacR is underscored by the 37 Å center-to-center distances between the recognition helices of each QacR dimer both in its DNA bound and apo (drug-free) form (M.A.S. & R.G.B, unpublished results) compared to a distance of only 34 Å for proteins that bind consecutive B-DNA major grooves. The identical center-to-center distances and the DNA contacts of each QacR dimer suggest that binding of the first dimer is the energetically costly step, whereby the IR1 site undergoes significant reconfiguration to produce the optimal QacR–DNA major groove interaction. Key to cooperativity, a DNA conformation is created that can bind the second QacR dimer readily and stabilize the IR1 site in an unwound state.

3. The QacR-rhodamine 6G structure and drug binding stoichiometry The binding of an array of structurally diverse, cationic lipophilic compounds to QacR results in its induction and the resultant expression of the qacA (or qacB) gene(s) [8]. To understand the mechanism of induction of QacR and gain insight into the intriguing phenomenon of multidrug recognition, the crystal structures of QacR bound to six “drugs”, including rhodamine 6G (R6G), ethidium (Et), dequalinium (Dq), crystal violet (CV), malachite green (MG) and berberine (Be) were determined [28] (Fig. 3). The first structure determined was QacR bound to R6G (Fig. 1). This structure revealed, unexpectedly, that QacR binds one R6G molecule per dimer. Equilibrium dialysis and Scatchard analyses confirmed the 1:2 (drug:QacR subunit) stoichiometry. Interestingly, this stoichiometry reflects another departure of QacR

2.4. QacR and TetR: Structural homology does not necessarily equate with functional homology QacR and TetR share structurally homologous DNA binding motifs and both insert their HTH motifs deeply into the DNA major groove. Neither QacR nor TetR contacts the DNA minor groove [21,29]. The recognition helix of each protein is unusually short but nearly maximally utilized in DNA interactions; of the seven residues of this helix, six are engaged in DNA binding in both QacR and TetR. However, these features represent the only commonalities to the DNA recognition modes of these two family members. Indeed, TetR and QacR ultimately arrive at specificity in DNA binding by quite distinct mechanisms. Specifically, TetR, recruits Arg28, located outside its recognition helix, to make a base pair specific contact [21]. In contrast, QacR does not employ residues outside of α3 but takes a more extreme approach to ensure DNA binding specificity and affinity through the cooperative binding of two dimers [29]. Another significant difference between these two protein–DNA complexes is the conformation of the bound DNA. TetR kinks its binding site and induces a 17◦ bend towards the protein to optimize the position of its HTH motifs for specific base interactions within each DNA half site. This DNA distortion also includes localized major groove widening. QacR, on the other hand, widens the major groove of the entire IR1 binding site smoothly and bends its DNA site by only 3◦ . These distinctions are reflected in the dramatically different HTH center-to-center distances observed in QacR (37 Å) and TetR (31 Å). Thus, an important lesson derived from comparisons of the QacR–DNA and TetR–DNA structures is that even structurally homologous proteins of the same family that share a similar function, i.e., repression, can utilize quite different mechanisms of action. The structure of one family member is unlikely to describe all members properly.

Fig. 3. Six QacR–drug complexes. Crystals and close up of the drug binding pockets of the corresponding six QacR–drug complexes. The top of each structure is the corresponding crystal. In the close up of the binding pockets, the complexes are color-coded appropriately to match the “drug color” with the exception of the Dq structure (a colorless drug), which is colored “clear” blue. For the sake of clarity only key residues, including the acidic residues (colored red) that neutralize the positive charges of each drug are shown. The carbon, nitrogen and oxygen atoms of the drugs are colored white, blue and red, respectively. (A) QacR–R6G complex. (B) QacR–Et complex. (C) QacR–Dq complex. (D) QacR–CV complex. (E) QacR–MG complex. (F) QacR–Be complex.

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from family member TetR [12], which binds two tetracycline molecules per dimer. 3.1. The QacR drug induction mechanism: Tyrosine expulsion The high resolution structures of both the DNA-bound and drug-bound forms of QacR allowed the detailed description of its drug induction mechanism [28,29]. Comparison of the R6G-bound and IR1-bound QacR structures reveals that drug binding triggers a coil-to-helix transition of residues Thr89–Tyr93 of the drug-bound subunit, such that the C-terminus of α5 is elongated by a turn; the drug-free subunit of the dimer retains the “DNA-bound” coil structure (Fig. 4). Likely favoring this transition is the conversion A

B

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of Tyr92, the only Ramachandran outlier of DNA-bound QacR, to a favorable conformation. When in its unfavorable coil position, Tyr92 plays a central role in formation of the protein hydrophobic core, which is exceptionally rich in aromatic residues and includes, in addition to Tyr92, the side chains of residues Trp61, Trp65, Phe79, Tyr82, Tyr93, Tyr123, Tyr127, Phe131, Trp140, Phe178 and Phe182. Upon drug binding Tyr92, as well as Tyr93, are expelled from the hydrophobic core, Tyr92 is removed to the solvent while Tyr93 is translocated to a peripheral site where it stacks with the R6G drug (Fig. 4). Because the drug-binding pocket is produced by ejection of Tyr92 and Tyr93 from the core and the concomitant elongation of α5, these tyrosine side chains play the essential role of structural drug surrogates, stabilizing the inducer binding pocket in the absence of drug. The coil-to-helix transition is required not only for drug binding but is also the key to the induction process. Indeed, the formation of the additional turn of helix in α5 leads to the relocation of α6 and consequently its tethered DNA binding domain, causing a 9.1 Å displacement and 36.7◦ rotation of the DNA binding domain relative to its IR1-bound position (Figs. 1 and 4). Aiding this structural change is a concomitant pendulum motion within α4 that occurs upon drug binding (Figs. 1 and 4). These movements within the drug-bound subunit of QacR also cause structural changes in the drug-free subunit of the dimer whereby its DNA binding domain undergoes a 3.9 Å translation and 18.3◦ rotation compared to the DNA-bound conformation. The sum result of the conformational changes of both subunits is the increase of the center-to-center distance between the recognition helices from 37 Å (DNA-bound form) to 48 Å (drug-bound form). This induction mechanism contrasts with that of TetR, which binds two ligands per dimer and utilizes an induction mechanism that involves several “through helix” motions and in which there is only a 3 Å increase in the center-to-center distance of the recognition helices upon tetracycline binding [21]. 3.2. Drug entrance and QacR–R6G interactions

Fig. 4. QacR induction by the tyrosine expulsion mechanism. (A) Superimposition of the core drug-binding region, residues 55–188, of the DNA-bound conformation (yellow) onto the drug-bound conformation (blue) revealing the structural changes that occur upon drug binding. R6G is depicted as a red stick model. (B) Closeup of the R6G binding pocket before (yellow) and after (blue) drug binding depicting the drug-induced expulsion of Tyr92 and Tyr93 from the core and concomitant coil-to-helix transition, in which α5 is lengthened by one turn. Reprinted with permission from Schumacher et al. (2001) Science 294: 2158–2163. © American Association for the Advancement of Science.

The drug binding pocket created by tyrosine expulsion is extensive and composed of residues from all helices of the multidrug binding domain except, α9, as well as residues from α8 . The only apparent portal to the ligand binding site is formed by the divergence of α6, α7, α8 and α8 (Fig. 1). The relocation of the region from α8-turn-α9 of the drug-bound subunit towards the drug binding pocket of the neighboring drug-free subunit, which is caused by the movement within the conformational switch region of α5 and α6, greatly limits access to the drug-binding site of the other subunit and likely explains the observed 1:2 drug:QacR stoichiometry. As expected from the exceptionally aromatic nature of the QacR drug binding pocket, these residues play a key role in QacR–drug interactions. In the QacR–R6G structure, the three-ring system of R6G stacks between the side

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chains of Trp61 from α4 and Tyr93 from α5 (Fig. 3). The R6G phenyl moiety makes stacking and hydrophobic interactions with Tyr123 and Leu54 whereas its oxygen moiety, O27A, hydrogen bonds to a water molecule located in the internal binding pocket. As observed in all the QacR–drug complexes, solvent molecules occupy portions of the drug-binding pocket that are not occupied by drug. Another notable feature of the QacR drug binding site is the large number of polar residues located within the pocket, including several asparagine, glutamine, threonine and serine residues that add versatility for drug binding by contacting the polar moieties of different drugs. For example, in the R6G complex QacR residues Gln64 and Thr89 contact the R6G N2 and Gln96 contacts R6G N1. The R6G ring system is further secured by a contact between the R6G central ring O1 atom and the Oγ of residue Thr89. A distinctive feature of multidrug binding proteins that recognize cationic drugs is the presence of one or more buried glutamates or aspartate residues. The importance of such buried acidic residues was revealed in studies on MDR transporters, which recognize cationic lipophilic drugs. In these proteins, glutamate or aspartate residues are often located on membrane embedded transmembrane helices and have been shown to be essential for transport function [7,19,22]. Direct evidence that acidic residues are involved in drug binding was first demonstrated by the structure of the ternary complex of the MerR family member, BmrR bound to the drug TPP+ and the bmr promoter [34]. In BmrR the acidic residues Glu253, Glu266 and Asp47 , from the adjacent subunit, are all within ∼8 Å of the formal charge of the bound TPP+ although only Glu253 is essential for drug binding [32]. In the QacR drug-binding site there are four glutamate residues, Glu57, Glu58, Glu90 and Glu120, all of which are at least partially buried and line the drug binding pocket roughly equidistant from the center of the large pocket. Glu57 and Glu58, on helix α4, and Glu120 on helix α7, are located at the solvent– protein interface and their side chains need only to rotate to reach into the pocket to interact with drug. However, residue Glu90, which is directly involved in the coil-to-helix transition of α5, is relocated into the pocket only upon drug binding. Indeed, in the QacR–R6G complex it is Glu90, that neutralizes the positively charged ethyl ammonium group of the drug (Fig. 3).

Fig. 5. The multisite model for multidrug binding and recognition. Superimposition of drug binding pockets of the QacR–R6G, QacR–Et and QacR–Dq complexes, which highlight the multisite binding pocket of QacR. R6G, Et and Dq are colored red, orange and dark blue, respectively.

plex reveals the same coil-to-helix transition in the α5 Cterminus, i.e., the same induction mechanism as described by the QacR–R6G structure. In fact, all QacR–drug complex structures determined thus far reveal the identical induction mechanism. A second significant feature of the QacR–Et complex is that, unlike the R6G complex, the positive charge on the Et is not complemented by Glu90, but by one of the other four acidic residues that line the pocket, Glu120 (Fig. 3). However, as observed in the QacR–R6G complex, aromatic and polar contacts are abundant in the QacR–Et complex. Two aromatic residues, Tyr103 and Phe162 from the other subunit (Phe162 ) sandwich the Et phenanthridinium ring system and additional stacking interactions between Tyr123 and the exocyclic 6-phenyl moiety of Et, anchor this cytotoxin. The most solvent-exposed Et phenanthridinium ring stacks with Tyr107 while the more buried phenanthridinium phenyl group is wedged between the side chains of Ile99 and Ile100 with its N1 amino nitrogen engaged in a hydrogen bond with the Oε2 of Gln96.

3.3. QacR–ethidium structure: A second binding site 3.4. Dequalinium binding confirms multisite binding The structure of QacR bound to a second drug, ethidium, revealed an unexpected and striking finding, the presence of a second binding pocket within the large QacR drug binding pocket. This “Et pocket” is distinct but partially overlaps that of the “R6G binding pocket” (Figs. 3 and 5). The Et pocket is closer to the surface than the R6G pocket, however the phenanthridinium ring system of the bound Et is successfully inserted to elicit the tyrosine expulsion process and therefore, flip the induction switch. Indeed, the Et com-

The discovery of two separate but partially overlapping binding sites in the extended drug-binding pocket of QacR is striking. The volume of this drug-binding pocket, which has dimensions of ∼10 Å × 9 Å × 23 Å, is 1100 Å3 whereas the largest pocket of drug-free QacR is under 400 Å3 [6]. Computational analysis of QacR predicted the top two binding sites for ligands as corresponding to the R6G and Et pocket, thus providing additional support for the existence of

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two separate, but linked binding sites within the single large pocket [6]. The presence of the R6G and Et “mini” drug binding sites was interesting because in addition to binding monovalent lipophilic cations, QacR binds and is induced by bivalent cationic lipophilic compounds, e.g., dequalinium (Dq). If, in fact, there are two drug-binding sites in QacR, the inducer Dq, which contains two positively charged 4-amino-2-methylquinolinium moieties connected by a decamethylene linker, should bind both pockets. This prediction was fulfilled and visualized by the 2.54 Å resolution structure of the QacR–Dq complex, which revealed that the positively charged aminomethylquinolinium moieties bind in the two QacR pockets defined as the R6G pocket and the Et pocket (Figs. 3 and 5). The decamethylene linker of Dq is held in place by multiple van der Waals contacts with the side chains of residues Leu54, Ile99, Met116 and Leu119. In the R6G pocket, the quinolinium group is sandwiched between Trp61 and Tyr93, similar to R6G binding. However, unlike R6G, the positively charged nitrogen moiety of this quinolinium group is not neutralized by Glu90 but by Glu57 and Glu58 (Fig. 3). In the Et pocket the Dq contacts are homologous to those observed in the QacR–Et complex in that the quinolinium group is sandwiched between Tyr103 and Phe162 and its positively charged nitrogen is complemented by Glu120 (Figs. 3 and 5). 3.5. The natural product berberine binds QacR The identification of the natural substrates of multidrug binding proteins is a subject of intense study. Because bacteria are associated with mammals, most of which consume plants harboring significant levels of amphipathic, cationic alkaloids, it has been suggested that MDR transporters evolved in response to bacteria being exposed to these alkaloids [15]. Indeed, the preferred substrates of most multidrug efflux proteins are hydrophobic cations. Of relevance, the plant alkaloid, berberine (Be), has been shown to be a substrate of QacA and is a potent antimicrobial in the absence of MDR transporters [15]. Recently, Be was demonstrated to induce QacR and therefore, represents the only known natural product to do so. To examine the binding of this “natural drug” to QacR, the structure of the QacR–Be complex was determined [28]. The resulting structure provides another example of a compound that binds within the R6G pocket of QacR (Fig. 3). Like R6G, the Be anthracene ring system stacks between Trp61 and Tyr93 while the 1,3-dioxa-6aazoniaindeno group interacts with Tyr123 and makes hydrogen bonds from its 1,3 oxygens to the side chains of Asn157 and Glu120. At the opposite end of the ring system, the two 8,9-dimethoxy groups reach into a solvent exposed region that is formed when the coil-to-helix transitional switch is thrown. Lastly, the positive charge, centered on the N1 nitrogen of berberine (Fig. 3), interacts with the side chains of Glu57 and Glu58 in a manner comparable to that of the dequalinium quinolinium nitrogen bound in the R6G pocket.

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3.6. QacR–crystal violet and malachite green complexes: Non-planar drugs In addition to planar drugs such as R6G, Et and Dq, QacR is also induced by compounds such as the dye crystal violet (CV), which has a rounded propeller-like geometry. Inducers of this type cannot sandwich into either the R6G or the Et binding pockets of QacR. The structure of the QacR–CV complex reveals that CV binds in the area that overlaps the R6G and Et pockets such that its aryl moieties make contacts to hydrophobic residues at the edges of each of the pockets (Fig. 3). In this binding mode, the methyl groups of one dimethylaminophenyl moiety make hydrophobic interactions with Phe93 and Trp61 of the R6G binding site, while the phenyl group stacks against Tyr123. The methyl and phenyl groups of a second dimethylaminophenyl moiety are within van der Waals distance of Tyr103 and Phe162 , i.e., the Et pocket. The CV methyl groups also engage in hydrophobic interactions with Ile100 and Ile100 . Finally, the third dimethylaminophenyl moiety is anchored to a pocket between α7 and α8 where it contacts Ala153, Glu120, Asn154 and Asn157. This distinctive binding mode permits the stabilization of the delocalized positive charge of CV (Fig. 3) via interactions between two of its amino groups and Glu90 and Glu120. Given this mechanism of charge neutralization by both Glu90 and Glu120, it was predicted that replacement of one of the three dimethyl amino groups with a hydrogen, as found in malachite green (MG), should not alter binding dramatically. Indeed, the QacR–MG structure revealed that MG binds in essentially the same pocket as CV and its positive charge is neutralized in the same manner as observed for CV (Fig. 3).

4. The multisite model for multidrug recognition The structures of the QacR–drug complexes provide the first atomic view of any multidrug binding protein bound to several of its structurally diverse ligands. These structures reveal several features that likely serve as the basis for understanding multidrug recognition by other multidrug-binding proteins, in particular MDR transporters. First, QacR exhibits an expansive drug binding site with several chargeneutralizing residues lining and surrounding the pocket. In QacR, these four glutamate residues, Glu57, Glu58, Glu90 and Glu120, are nearly equally spaced around the pocket with an average distance of ∼4 Å from their carboxylate oxygens to the formal positive charge of their ligands (Fig. 3). Similar charge-charge neutralization is expected to be used by the MDR transporters. Specifically, several MDR transporters including QacA, which effluxes mono and bivalent cationic lipophilic drugs, are predicted to have more than one buried charged residue as a component of their multidrug binding pockets [22]. The recent structure of the E. coli resistance-nodulation-cell division (RND) multidrug transporter, AcrB, which was solved in the absence

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of drugs, corroborates this hypothesis by revealing the presence of charged residues that are buried in the transmembrane domain [18]. Second, the pocket contains an impressive number of aromatic and polar residues, which can act in a drug-dependent manner as either hydrogen bond donors or acceptors. The importance of polar residues in multidrug binding is also seen in the structure of the human MDR transcription factor PXR bound to SR12813. In this structure multiple orientations of the SR12813 drug are stabilized by a different complement of polar side chains [33]. Finally and perhaps the most critical feature of the multidrug binding mechanism revealed by the six QacR–drug structures is the presence of several, separate, but linked binding sites within one extended, multifaceted drug binding pocket. The multisite multidrug binding mechanism utilized by QacR is consistent with and provides further insight into accumulating data, that indicate the presence of multiple binding sites in both secondary and ATP-dependent multidrug transporters [13,14,17,22,24,30,31]. The most studied of these transporters is P-glycoprotein, for which a two-site model was postulated over a decade ago on the basis of kinetic analysis of drug stimulated ATPase activity. Those studies demonstrated that cyclosporin A competitively inhibits verapamil stimulated ATPase activity whereas daunorubicin, gramacidin D, vinblastine and colchicine result in allosteric inhibition [30]. Noncompetitive inhibition of R6G transport by quinine, puromycin and colchicine has been documented for the yeast MDR transporter Pdr5p [13] and van Veen et al. have shown that nicardipin interacts with the lactocococcal multidrug transporter LmrP at a binding site distinct from but allosterically linked to the vinca alkaloid binding site [24]. Similarly, competition studies have provided strong support for two distinct drug-binding sites in QacA [22] and recently the E. coli MDR transporter, MdfA, was shown to bind TPP+ and chloramphenicol simultaneously in apparently distinct, but interacting sites [14]. Thus, these biochemical and kinetic studies strongly suggest that the presence of two or more overlapping or linked drug binding sites is a general feature of multidrug binding. The QacR–drug structures provide structural insight into how such multidrug recognition and binding can occur.

5. Conclusions The high resolution structures of QacR bound to six structurally diverse drugs has provided an atomic view of the phenomenon of multidrug binding. These structures reveal a number of features that will likely be shared by cationic drug effluxing MDR pumps and include sizeable drug binding pockets, which are exceptionally rich in aromatic residues and contain one or more charge neutralizing acidic residues. The QacR–drug complex structures unveiled the presence of multiple drug binding “mini-pockets” within a larger pocket, thereby providing a structural context for drug-binding data that have accumulated for MDR transporters over the past

20 years. Indeed, a series of proximal minipockets with their unique complement of residues offers a ready explanation for the binding of charged and uncharged drugs. However, the precise mechanisms by which anionic and non-charged ligands are recognized by MDR proteins is still unresolved at the atomic level and may abide by different rules as suggested by the structure of the regulator MarR bound to salicylate [2,3]. This structure showed that the anionic salicylates are not bound in large pockets, but in solvent exposed crevices [3]. Furthermore, although the salicylate binding sites in MarR contain charge neutralizing arginines, they possess no aromatic residues. Clearly, additional structures of multidrug binding, gene regulators are needed to understand more fully not only the mechanism of multidrug recognition and binding but to unveil the gene regulatory mechanisms utilized by these proteins.

Acknowledgements The authors would like to thank the Burroughs Wellcome Trust (to M.A.S.) and the National Institutes of Health (AI48593 to R.G.B.) for financial support. We would also like to thank Drs. Melissa Brown and Ron Skurray, for their insightful discussions on the phenomenon of multidrug binding and Mr. Marshall Miller for biochemical analyses of QacR drug binding.

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