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Structural Mechanisms of QacR Induction and Multidrug Recognition

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Science  07 Dec 2001:
Vol. 294, Issue 5549, pp. 2158-2163
DOI: 10.1126/science.1066020

Abstract

The Staphylococcus aureus multidrug binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by structurally diverse cationic lipophilic drugs. Here, we report the crystal structures of six QacR-drug complexes. Compared to the DNA bound structure, drug binding elicits a coil-to-helix transition that causes induction and creates an expansive multidrug-binding pocket, containing four glutamates and multiple aromatic and polar residues. These structures indicate the presence of separate but linked drug-binding sites within a single protein. This multisite drug-binding mechanism is consonant with studies on multidrug resistance transporters.

The emergence of multidrug resistance (MDR) has been attributed in part to membrane transport systems capable of effluxing a broad spectrum of toxic compounds (1–4). In cancer cells, resistance to chemotherapeutic agents is mediated by the P-glycoprotein efflux pump (5, 6), whereas in bacteria MDR transporters are responsible for resistance to many clinically important antimicrobial compounds (7, 8). The serious health threat posed by the emergence of strains of antibiotic-resistant Staphylococcus aureus appears to have been exacerbated by plasmid-encoded MDR efflux pumps such as QacA, which confer resistance to monovalent and bivalent cationic lipophilic antiseptics and disinfectants such as quaternary ammonium compounds (QACs) (9–12). Studies on MDR transporter proteins have provided evidence for the presence of multiple, possibly overlapping, drug-binding sites within each protein (13–18). However, advances in understanding the basis of multidrug recognition has been hampered by the difficulty of performing high-resolution structural analyses on integral membrane proteins.

Structural studies on cytosolic multidrug-binding regulatory proteins provide an alternative in delineating multidrug binding mechanisms. Structures of several regulatory proteins bound to a particular drug have been determined; the Escherichia coliMarR repressor bound to salicylate (19), the Bacillus subtilis transcriptional activator BmrR bound to tetraphenylphosphonium (TPP+) and DNA (20) and the human nuclear xenobiotic receptor (PXR) bound to the cholesterol lowering drug SR12813 (21). The 23-kDS. aureus QacR repressor, a member of the TetR family of repressors (22), is another multidrug binding protein, which regulates the expression of the qacA MDR pump gene (23, 24). QacR is induced by binding mono and bivalent cationic lipophilic drugs, many of which are substrates of QacA. We report here the structures of QacR bound to six of these structurally diverse cytotoxic agents (“drugs”), rhodamine 6G (R6G), ethidium (Et), dequalinium (Dq), crystal violet (CV), malachite green (MG) and berberine (Be) (Fig. 1).

Figure 1

The chemical structures of six cationic lipophilic drugs bound by QacR that were used in this study. The positively charged nitrogens are indicated. Note the bivalent nature of the inducer, dequalinium.

The structure of QacR bound to R6G was determined by multiple wavelength anomalous dispersion (MAD) (Fig. 2A, Table 1) [Web fig. 1, Web table 1 (25)] (26, 27). QacR is entirely helical and comprises nine helices (Fig. 2B). The first three helices form a three-helix bundle DNA binding domain, which contains a helix-turn-helix (HTH) motif (α2 and α3). Helices 4 through 9 form the drug binding/dimerization domain and formation of the dimer buries 1530 Å2 of surface area per monomer. The structure reveals that QacR binds one R6G molecule per dimer (Fig. 2B). Equilibrium dialysis and Scatchard analyses [Web fig. 2 (25)] confirmed the 1:2 (drug:QacR subunit) stoichiometry (28). In contrast, the family member TetR binds two tetracycline molecules per dimer. However, the multidrug binding proteins, EmrR and BmrR, also appear to bind their ligands with a 1:2 (drug:subunit) stoichiometry (29, 30).

Figure 2

The mechanism of multidrug binding and induction. (A) Simulated annealing omit map of the R6G drug-binding pocket. Composite electron density omit map (contoured at 1.5 σ) calculated with a starting temperature of 2000 K and excluding R6G from the model. Carbon, nitrogen, and oxygen atoms are colored yellow, light blue, and red, respectively. (B) Structure of the drug-bound QacR-R6G complex. QacR consists of nine helices: α1(3-18), α2(25-32), α3(36-42), α4(46-71), α5(75-88; 75-93 in the drug-bound subunit, see below), α6(96-108), α7(110-136), α8(145-162), and α9(168-185). The yellow region (residues 89 to 93) forms a helix upon drug binding. The bound R6G is shown as balls and sticks with carbon, oxygen, and nitrogen colored gray, red, and blue, respectively. (C) Superimposition of the core drug-binding region, residues 55 to 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. The DNA-binding recognition helix is labeled Hr. (D) Close-up view of the R6G binding pocket before (yellow) and after (blue) drug binding depicting the drug-induced tyrosine expulsion of Tyr92 and Tyr93 from the core and concomitant coil-to-helix transition in which residues 89 to 93 are incorporated into α5, thus lengthening it by one turn.

Table 1

QacR-drug complex data collection and refinement.

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Comparison of the R6G-bound structure with a QacR-DNA structure determined in our lab (Fig. 2, C and D) reveals that binding of the drug triggers a coil-to-helix transition of residues Thr89-Tyr93 (Fig. 2B) of the drug-bound subunit only, such that the COOH-terminus of α5 is elongated by a turn; the drug-free subunit in the dimer retains the “DNA-bound” coil structure (31) [Web fig. 1 (25)]. This transition is likely facilitated by the conversion of Tyr92, which is the only Ramachandran outlier in the drug-free state, from an unfavorable to a favorable conformation. In its unfavorable coil position, Tyr92 plays an essential role in formation of the protein hydrophobic core. Upon drug binding, Tyr92 is expelled from the hydrophobic core into the solvent. Tyr93 is also dislodged from the hydrophobic core to a peripheral site, where it stacks with R6G (Fig. 2, C and D). Tyr92 and Tyr93 thus act as structural drug surrogates that stabilize the inducer-binding pocket in the absence of drug.

The coil-to-helix transition is the key not only for drug binding but also for induction as the formation of the additional turn of helix leads to the relocation of α6 and its tethered DNA-binding domain (Fig. 2C). Movement of α6 leads to a 9.1 Å translation and 36.7° rotation of the DNA-binding domain, relative to DNA-bound QacR (31). There is also a pendulum motion of α4 upon drug binding (Fig. 2C). Concomitant structural changes, necessary for retention of interaction between the α6 and α6′ helices, in the drug-free subunit of the dimer also occur. The DNA-binding domain of the drug-free subunit undergoes a 3.9 Å translation and 18.3° rotation compared to the DNA-bound conformation. Overall, there is a large increase in the center-to-center distance of the recognition helices from 37 Å (DNA-bound form) to 48 Å (drug-bound form). In contrast, in TetR there is only a 3 Å increase in the center-to-center distance of the recognition helices upon tetracycline binding (32).

The drug-binding pocket created by tyrosine expulsion is extensive and composed of residues from all helices of the inducer-binding domain except α9, as well as residues from α8′ (where prime indicates the other subunit of the dimer). The portal to the aromatic ligand-binding site is formed by the divergence of α6, α7, α8, and α8′. Because this portal is the only apparent entry into the pocket, the structure suggests a possible explanation for the 1:2 drug:QacR stoichiometry. The COOH-terminus of α8, the intervening turn (T) and the NH2-terminus of α9 are apposed to residues in the conformational switch region of α5 and α6 (Fig. 2C), and thus the drug induced coil-to-helix transition forces the movement of α8-T-α9 into the drug-binding pocket of the neighboring subunit, limiting access to its entrance.

Within the drug-binding pocket, the three-ring system of R6G is wedged between Trp61 from α4 and Tyr93 from α5 (Fig. 3A). The phenyl moiety makes hydrophobic interactions with Tyr123 and Leu54, whereas its oxygen moiety, O27A, hydrogen bonds to a water molecule. Additional water molecules fill the portion of the large drug-binding pocket that is unoccupied by drug. Asparagine, glutamine, threonine, and serine residues in the pocket provide versatility for contacting polar moieties of different drugs. In the QacR-R6G complex, Gln96 contacts the R6G N1 atom and Gln64 and Thr89 contact the R6G N2. The R6G ring system is further anchored by an interaction between the central ring O1 atom and the Oγ of Thr89.

Figure 3

Multidrug recognition by QacR. For the sake of clarity, only key residues are shown (no solvent) including the acidic residues (colored red) that neutralize the positive charges of each drug. 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. The distances between the positively charged nitrogens of each drug and QacR acidic residues are: R6G [Oɛ2(Glu90)-N1(R6G), 3.98 Å]; Et: [Oɛ2(Glu120)-N2(Et), 3.95 Å]; Dq: [Oɛ1(Glu57)-N2(Dq), 4.75 Å; Oɛ1(Glu58)-N2(Dq), 4.93 Å; and Oɛ1(Glu120)-N1(Dq), 4.80 Å]; CV: [Oɛ1(Glu90)-N2(CV), 3.96 Å; Oɛ2(Glu120)-N3(CV), 3.85 Å]; MG: [Oɛ1(Glu90)-N2(MG), 3.35 Å; Oɛ2(Glu120)-N3 (MG), 3.60 Å]; Be: [Oɛ1(Glu57)-N1(Be), 5.64 Å; Oɛ1(Glu58)-N1(Be), 4.93 Å].

A distinguishing feature of multidrug binding proteins that recognize cationic drugs appears to be the presence of buried negatively charged glutamates or aspartate residues. This was clearly demonstrated in the BmrR-TPP+-DNA structure, which contains a buried glutamate, Glu253, critical for drug binding (20). In the QacR drug-binding site there are four glutamate residues, Glu57 and Glu58 from α4, Glu90from α5, and Glu120 from α7, all of which are partially buried and surround the drug-binding pocket. In the QacR-R6G complex, Glu90 neutralizes the positively charged ethyl ammonium group of the drug (Fig. 1). Glu90 is also part of the conformational switch region of α5 and is translocated into the drug-binding pocket only upon drug binding.

All QacR-drug structures described in this study (Fig. 1 and Table 1) utilize the identical induction mechanism. In the QacR-Et structure the Et is bound in a pocket that is distinct but partially overlaps that of the R6G binding site (compare Fig. 3, A and B). The “Et pocket” is closer to the proposed “portal” entrance than the “R6G pocket,” yet the phenanthridinium ring system of the Et is adequately inserted to elicit the tyrosine expulsion process and thus, flip the induction switch. Unlike the R6G complex, the formal positive charge on the Et (Fig. 1) is not complemented by Glu90, but by Glu120 (27). Two aromatic residues, Tyr103 and Phe162′, sandwich the phenanthridinium system and additional stacking interactions, including those between Tyr123 and the ethidium exocyclic 6-phenyl moiety, secure the Et (Fig. 3B). The most buried phenanthridinium phenyl group is wedged between Ile99 and Ile100from α6, while its N1 amino group nitrogen is hydrogen bonded to the Oɛ2 of Gln96. As in the QacR-R6G, several solvent molecules occupy the void in the pocket that is not filled by drug.

The volume of the drug-binding pocket, which has dimensions of ∼10 Å by 9 Å by 23 Å, is 1100 Å3 as determined by the program “putative active sites with spheres” (PASS) (33), whereas the largest pocket of drug-free QacR is under 400 Å3 (Fig. 4, A and B). When PASS was used to search for potential ligand binding within the drug-removed QacR structure, the top two predicted binding sites, which have overlapping volumes, correspond to the centers of the Et and the R6G drug-binding pockets (Fig. 4A) (33). This computational analysis, therefore, provides additional unbiased support for the presence of two separate but potentially overlapping binding sites within a single pocket. In addition to binding monovalent lipophilic cations, QacR also binds bivalent cationic lipophilic compounds, e.g., dequalinium (Dq). The 2.54 Å resolution structure of the QacR-Dq complex reveals that the two positively charged aminomethylquinolinium moieties bind the pockets defined as the R6G pocket and the Et pocket, while the decamethylene linker adjusts to provide an optimal fit (Fig. 3C and Fig. 4A). In the R6G pocket, one quinolinium group is sandwiched between Trp61 and Tyr93, similar to R6G binding. However, unlike R6G, the positively charged nitrogen of this moiety (Fig. 1) is complemented by Glu57 and Glu58. In the Et pocket, the Dq contacts are functionally identical to those observed in the QacR-Et complex; the second quinolinium group is bound between Phe162′ and Tyr103, and its positively charged nitrogen is neutralized by Glu120 (Fig. 3C). The decamethylene linker is well ordered and makes multiple van der Waals contacts with the side chains of Leu54, Ile99, Met116, and Leu119.

Figure 4

The extended multisite binding pocket of QacR. (A) Superimposition of drug-binding pockets of the QacR-R6G, QacR-Et, and QacR-Dq complexes highlighting the multisite binding pocket of QacR. R6G, Et, and Dq are colored pink, orange, and yellow, respectively. The two top binding-site centers predicted from PASS (33) are depicted as blue balls and labeled ligand binding site 1 and 2 (LBS1 and LBS2). (B) Ribbon diagram of the drug-bound QacR dimer looking down from the “top” of the dimer. The drug-bound subunit is colored dark blue and the other is cyan. The binding-site volume calculated from PASS with drugs removed is depicted as a transparent surface. Shown within this volume as sticks are the drugs from all structures where R6G is pink, Et is orange, Dq is light yellow, MG is green, CV is violet, and Be is dark yellow. Note the optimal fit of the drugs within the extended binding site. The helices in the drug-bound protomer are labeled.

Unlike R6G, Et and Dq, the QacR inducer crystal violet (CV) (21) does not have a planar ring system (Fig. 1). Instead, this dye has a propeller-like geometry with dihedral angles between aryl groups and the central coordination plane of 27.7° (34). As a result, CV should not be able to sandwich into either the R6G or the Et binding pockets of QacR. The structure of the QacR-CV complex reveals that, somewhat like Dq, CV binds in the overlapping region between the R6G and Et pockets such that its aryl groups interact with hydrophobic residues at the edges of each pocket (Fig. 3D). Specifically, the methyl groups of one dimethylaminophenyl moiety make hydrophobic interactions with Tyr93 and Trp61 near the R6G binding site, whereas its 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., near 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 unique binding mode allows the dissipation of the delocalized positive charge of CV (Fig. 1) via interactions between two of its amino groups and glutamates, Glu90 and Glu120.

From the manner in which the delocalized charge of CV is neutralized by Glu90 and Glu1120, we predicted that replacement of the third dimethyl amino group with a hydrogen, as found in malachite green (MG), although removing several hydrophobic contacts, should not prevent binding. Indeed, the QacR-MG structure demonstrates that this dye binds in essentially the same pocket as CV, and its positive charge is similarly complemented as in CV (Fig. 3E). However, superimposition of the QacR CV and MG complexes reveals a slight shift in the position of one drug relative to the other, allowing each to make optimal van der Waals contacts with QacR residues, again underscoring the versatility of the pocket. Although MG binds QacR, whether it is a physiologically relevant inducer ofqacA is unknown.

The plant alkaloid, berberine (Be), is a substrate of QacA that induces QacR and is a potent antimicrobial in the absence of MDR transporters (35, 36). Be binds within the R6G pocket of QacR (Fig. 3F), and like R6G, the Be anthracene ring system is sandwiched between Trp61 and Tyr93 while the 1,3-dioxa-6a-azoniaindeno group stacks with Tyr123 and is anchored by hydrogen bonds from its 1,3 oxygens to Asn157. At the other end of the ring system, the two 8,9-dimethoxy groups extend into a solvent exposed region, which is formed when the coil-to-helix transitional switch is thrown. Finally, the positive charge, centered on the Be N1 nitrogen (Fig. 1), is surrounded by the side chains of Glu57 and Glu58 in a manner analogous to that observed for one of the aminomethylquinolinium nitrogens in the QacR-Dq structure.

In conclusion, QacR exhibits an expansive drug-binding site with four charge-neutralizing residues that line and surround the pocket. There is a large number of aromatic residues and several polar residues in the pocket that can act in a drug-specific manner as either hydrogen bond donors or acceptors. The importance of polar residues for multidrug recognition was demonstrated by the PXR-SR12813 structure in which multiple orientations of the SR12813 ligand were observed within its large binding pocket, and stabilized by a different complement of polar residues (21). In addition, the recent structure of the E. coli MsbA, a homolog of the multidrug ABC transporters, revealed a large putative ligand binding cavity lined by polar residues (37). Finally, the combined QacR-drug structures reveal several, separate, but linked binding sites within one extended and thus, multifaceted drug binding pocket. This is likely to be a crucial feature in multidrug recognition. Indeed, the use of such a multifaceted pocket is consistent with accumulating data indicating the presence of multiple binding sites in both secondary and ATP-dependent multidrug transporters (13–18).

  • * To whom correspondence should be addressed. E-mail: brennanr{at}ohsu.edu

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