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Role of aromatic and negatively-charged residues of DrrB in multi-substrate specificity conferred by the DrrAB system of Streptomyces peucetius Kenneth Brown, Wen Li, and Parjit Kaur Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01155 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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Role of aromatic and negatively-charged residues of DrrB in multi-substrate specificity conferred by the DrrAB system of Streptomyces peucetius This work was funded in part by the National Institutes of Health grant RO1 GM 51981-09.

Kenneth Brown, Wen Li, and *Parjit Kaur Department of Biology Georgia State University Atlanta, GA, 30303

*Corresponding author [email protected] Tel: 404-413-5405 Fax: 404-413-5301

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Abbreviations and Textual Footnotes Abbreviations: Transmembrane domains (TMDs); Nucleotide binding domains (NBDs); Transmembrane helices (TM helices); Doxorubicin (Dox), ethidium bromide (EB), multispecificity, multi-drug resistance


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Abstract

Resistance to the anticancer antibiotics, doxorubicin and daunorubicin, in the producer organism Streptomyces peucetius is conferred by an ABC transporter made of two proteins DrrA and DrrB, which together form a dedicated exporter for these two antibiotics. Surprisingly, however, the DrrAB system exhibits broad substrate specificity overlapping with well-studied multidrug resistance transporters, including P-glycoprotein. Therefore it provides an excellent model to study molecular basis of multi-specificity in a prototype efflux system with the potential to unravel the origin and evolution of multidrug resistance. It has been speculated that multispecificity in multidrug exporters may be generally determined by the number and location of aromatic residues. Strategically placed negatively-charged residues may also be critical for binding of cationic lipophilic drugs. We selected thirteen aromatic and four negatively-charged residues based on their location in/near the predicted drug-binding pocket of DrrB for analysis. Indeed mutations in most tested residues inhibited doxorubicin efflux drastically. Interestingly, several mutants lost resistance to doxorubicin and verapamil simultaneously but retained resistance to Hoechst33342 and/or ethidium bromide suggesting the presence of overlapping as well as independent drug-binding sites in a common drug-binding pocket of DrrB. This study provides the first comprehensive analysis of residues involved in drug binding in a bacterial multidrug resistance protein of the ABC superfamily, and it shows strong similarity in the molecular mechanism of poly-specific drug recognition between DrrAB and Pgp. Taken together, we conclude that aromatic residue-based multidrug specificity is conserved across domains and over long evolutionary periods. The significance of these findings is discussed.


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Multidrug resistance (MDR) is a major clinical problem both for the chemotherapy of cancer and for treatment of pathogenic infectious diseases. It results from the action of specialized proteins that have broad specificity and are able to carry out energy-dependent efflux of structurally and functionally unrelated compounds directly from the membrane in the process conferring resistance to them1-5. Multidrug transporters are ubiquitous in nature and are powered by either the proton-motive force or by hydrolysis of ATP. Their mechanism of transport is different from the classical transporters which carry out transport of only a single substrate or a group of closely-related substrates from an aqueous compartment 6, 7. The best characterized MDR protein to date is the mammalian ABC (ATP Binding Cassette) protein P-glycoprotein (Pgp), which carries out efflux of hundreds of structurally unrelated hydrophobic and amphipathic compounds resulting in multidrug resistance in cancer cells 8, 9. MDR transporters found in several bacterial species and in lower eukaryotes also share broad and overlapping substrate specificity with Pgp 10-13 It has been previously speculated that the degree of multi-specificity of an export protein may correlate with the content and distribution of aromatic amino acids in its transmembrane (TM) helices 14. In particular, the ring structure of aromatic residues may provide surfaces compatible to the geometry of the ring-containing compounds or increase flexibility of the drug binding region, thus promoting the binding and transport of a variety of such molecules 14, 15. A large body of work available on mammalian Pgp indeed points to the existence of a large and flexible drug-binding cavity, which can accommodate multiple substrates simultaneously by interactions with aromatic and hydrophobic residues in different parts of the pocket 9, 16-20. Recent structural analyses of lower eukaryotic homologs, C. merolae CmABCB1 and C. elegans


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Pgp, also showed the involvement of aromatic and bulky hydrophobic residues in forming the drug cavity in these proteins 11, 12, 20. Of the bacterial MDR proteins, structures of secondaryactive transport protein AcrB and two regulatory proteins BmrR and QacR (which control expression of secondary-active transporters Bmr and QacAB) have been reported, which suggested that both aromatic and negatively-charged residues participate in drug-binding in these proteins21-25. The structures of a few bacterial MDR proteins of the ABC superfamily (including Sav1866, MsbA, and McjD) are also available 10, 26, 27. These structures were, however, obtained in the absence of drug ligands or in the presence of nucleotides, therefore they do not provide direct molecular insights about the residues involved in ligand binding. McjD structure was found to be in a novel outward-facing occluded state which defined a large binding cavity. Mutagenesis of one aromatic and two polar residues located in this cavity showed that they are important for binding of the MccJ25 peptide10. Overall, very limited information is available on the molecular mechanism of multisubstrate recognition in bacterial MDR systems especially of the ABC superfamily. Moreover, other important questions related to the origin of the phenomenon of multidrug resistance have also remained largely unanswered. Since most of the antibiotic and drug resistance genes found in clinical settings are assumed to have their origins in environmental bacteria 5, the study of prototype drug transport systems found in organisms that produce and export antibiotics may provide answers to the above questions. The present study is specifically focused on understanding the molecular mechanisms underlying drug recognition and transport by the ABC transporter DrrAB found in the soil bacterium Streptomyces peucetius. This organism produces two widely-used anticancer antibiotics doxorubicin (Dox) and daunorubicin (Dnr) 28, 29. The drrAB genes, embedded within the gene cluster for biosynthesis of these antibiotics, code for a


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dedicated exporter of these two closely-related drugs. Surprisingly, however, the DrrAB system not only exports Dox with high efficiency, but it can recognize and transport multiple unrelated compounds 30. This finding raises two very compelling questions. What is the molecular basis of multi-specific drug recognition in this prototype drug exporter? Is this mechanism related to the mechanism seen in the mammalian ABC protein Pgp and other studied MDR proteins described above? Understanding DrrAB is therefore important not only for elucidating the molecular basis of multi-specific drug recognition in this system but also for its potential to reveal the origins and evolution of poly-specificity. Most ABC proteins involved in transport functions consist of two nucleotide binding domains (NBDs) and two transmembrane domains (TMDs), however the organization of these domains varies among different ABC proteins 4, 31. For example, in Pgp all four domains (2 NBDs and 2 TMDs) are present in one large protein consisting of two similar but non-identical halves, each half containing a TMD made of six α-helices and an NBD 8. On the other hand, in the simpler DrrAB system, these domains occur on separate subunits. Two subunits each of DrrA (NBD) and DrrB (TMD) interact to form a tetrameric complex in the membrane 32, 33, which carries out ATP-dependent efflux of Dox and other unrelated compounds 30. The DrrA protein exhibits significant sequence homology with the highly conserved motifs in the NBDs of other ABC proteins, including mammalian Pgp and bacterial proteins Sav1866, McjD, and MsbA, suggesting the presence of a common mechanism for ATP binding in all ABC proteins. However, DrrB bears rather limited homology to the TMDs of these ABC proteins, which is consistent with the absence of identifiable conserved substrate-binding motifs in transport proteins in general unless they are close homologs linked directly through evolution. Note that the DrrAB system belongs to the DRR subfamily of Class 3 ABC proteins whereas the other


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well-studied MDR proteins mentioned here belong to various subfamilies of Class 1 ABC proteins 34, indicating that these systems are evolutionarily distinct. Based on the current knowledge of MDR proteins, however, we assume that the overall structure of the drug-binding cavity in an MDR protein and the amino acid make-up of the cavity may be more important than the linear sequence of the residues. To determine if aromatic and negatively-charged residues indeed contribute to forming the drug-binding pocket/cavity in the DrrAB system (as seen in Pgp and other studied MDR proteins), we focused on analysis of these residues in DrrB which contains twenty four aromatic and four negatively-charged residues in or near its TM helices 35. Of these, thirteen aromatic and all four negatively-charged residues were selected for analysis based on their location near the predicted drug-binding pocket in a homology model of DrrB. The targeted residues were mutated to create both conservative and non-conservative substitutions which were then characterized using functional analyses, including drug efflux and multi-drug resistance. The data presented in this article show that mutations in twelve aromatic residues predicted to be critical for drug binding resulted in drastically reduced Dox efflux activity. Substitutions of the four negatively-charged amino acids to either negative or positively-charged residues also resulted in a drastic effect on Dox efflux. Multidrug resistance profile of selected mutants allowed further identification of specific and overlapping residues involved in binding of different drugs. Overall the results reported in this study suggest that aromatic residues contributed by several TM helices in DrrB are involved in forming a common drug-binding pocket which exhibits significant overlap and flexibility in drug-binding residues. It is currently not known if the four negatively-charged residues found to be critical for Dox efflux in this study are directly involved in drug binding or if they play an indirect role in drug efflux and resistance.


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The present study provides the first comprehensive analysis of the molecular basis of multi-specificity in a bacterial multidrug ABC transporter found in a producer organism, and it demonstrates that the molecular basis of poly-specific drug recognition in DrrAB, Pgp, and other studied MDR systems is very similar. We conclude that the aromatic residue-based mechanism of poly-specificity in MDR proteins is very ancient, and it is conserved over large evolutionary periods, thus bringing us closer to understanding the origin of multidrug recognition.


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Experimental Procedures (Materials and Methods) Materials – Doxorubicin, verapamil, Hoechst 33342, ethidium bromide (EB), and ATP were purchased from Sigma-Aldrich. Protease inhibitor cocktail, Creatine kinase and Creatine phosphate were purchased from Roche Diagnostics. Bacterial Strains and Plasmids - The bacterial E. coli strains and plasmids used in this study are listed in Table 1. Strain or Plasmid

Genotype/ Description

Reference

supE44 supF58 hsdR514

36

Strain LE392∆uncIC

galK2 galT22 metB1 trpR55 lacY1 ∆ uncIC XL-1 Blue

recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac

N43

acrA1 lac str gal ara

37

Plasmids pSU2718

Cloning vector,

29

pDX101

drrAB in pSU2718

29

Table 1. Media and Growth Conditions - E. coli cells were grown in LB medium at 37°C. Chloramphenicol was added to the medium, where indicated, to give a final concentration of 20 µg/mL. M9 medium38 was used where indicated. Site-directed Mutagenesis of DrrB - A number of point mutations were made to the aromatic and


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negatively-charged residues in the drrB gene using the Strategene QuikChange multisite-directed mutagenesis kit (La Jolla, CA). The kit uses complimentary primers that incorporate the mutations at the correct position. pDX101 plasmid was used as the template, as described previously 33. Preparation of Inside-out Vesicles (IOVs) – IOVs were prepared by the protocol published previously 30. Briefly, E. coli LE392∆uncIC cells 36 containing each of the mutants mentioned above were grown in 0.4 L of LB medium containing 20 µg/mL chloramphenicol at 37°C until mid-log phase. Once reached, the cells were induced with 0.25 mM Isopropyl β-D-1thiogalactopyranoside and incubated for another 3 hours at 37°C. The cell pellet was then resuspended in 10 mL of 1x PBS buffer, pH 7.4, and supplemented with 1 mM 1, 4-dithiothreitol and one EDTA-free, protease inhibitor cocktail tablet. Cells were lysed twice using a French Press at 16,000 psi, and the lysates were centrifuged at 10,000 x g for 20 minutes at 4°C to remove unbroken cells. The membrane fraction was prepared and washed twice with 10 mL 1x PBS buffer as described previously 30. In Vitro Dox Efflux using IOVs - Dox fluorescence was observed on an Alphascan-2spectrofluorometer (Photon Technology International, London, Ontario, Canada) using an excitation wavelength of 480 nm and emission wavelength of 590 nm. The slit widths for both excitation and emission were set at 1.5 mm, and the data were collected every 0.1 seconds for a total of 400 seconds. The Dox efflux assay conditions described previously 30 were used. Briefly, 500 µg of the IOVs were suspended in 3 mL of 1x PBS buffer, pH 7.4 and supplemented with 0.1 mg/mL creatine kinase, 5 mM creatine phosphate, and 1 µM Dox. Fluorescence detection was started after Dox was added. After 100 seconds, the reaction was paused, and 1 mM MgSO4 , and 1 mM ATP, pH 7.5, were added and detection continued until 400 seconds.


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Efflux activity was determined by the slope of the linear portion of the chart immediately following the additions of MgSO4 and ATP (approximately the 100-200 second interval). Western Blot Analysis – 30 µg of membrane (IOV) protein was analyzed by electrophoresis on 12% SDS-polyacrylamide gels. This was followed by Western blot analysis using anti-DrrA or anti-DrrB antibodies 29. The intensity of bands was quantified using Multi Gauge V2.3 software. Drug Resistance Assays– Resistance to different drugs, including Dox, verapamil, Hoechst 33342, and EB was determined. E. coli N43 cells 37 containing wild type pDX101 or mutants were grown at 37°C in LB medium containing chloramphenicol to mid-log phase. 1:200 dilutions of the cultures were made in 3 mL M9 medium 38 containing different concentrations of each drug, followed by incubation with shaking at 225 rpm at 37°C for 16 hours. OD600 was then recorded for each of the samples. Note that the Mueller-Hinton broth was used (instead of M9 medium) for analysis of Hoechst 33324 resistance according to a previous report 39. Secondary Structure Predictions - Secondary structure predictions of DrrB were carried out using the Predictprotein server (predictprotein.org), which predicts transmembrane helices found within proteins. Analysis of the homology model of DrrB - A 3-D homology model of the wild type DrrB protein was previously created by using the online modeling software PHYRE 33, 40. Pymol v.1.7.4.5 was used to view the structure for analysis of aromatic residues found in TM helices of DrrB.


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Results Mutagenesis of DrrB – To determine if the DrrB protein exhibits significant sequence homology to the previously studied MDR proteins, including the mouse Pgp, C. elegans Pgp, CmABCB1, Sav1866, and McjD,10-12, 17, 26, and to determine if the previously identified drug-binding residues in these proteins are conserved in DrrB, a Clustal alignment of DrrB with the TMDs of these five ABC proteins was carried out (Fig. S1). Residues previously shown to be critical for drug interactions in mouse Pgp, C. elegans Pgp, CmABCB1, and McjD proteins are highlighted in the alignment. Fig. S1 shows that DrrB contains two highly conserved prolines and two glycines, which align with the corresponding conserved residues in the other TMDs (highlighted in green). This is consistent with the alignment of the ABC MDR proteins reported previously 12. Other than this conservation, however, DrrB exhibits very limited overall sequence identity with these five proteins (Figs. S1 and S2), therefore making it difficult to glean useful information about functional residues of DrrB. Note that the DrrA protein, on the other hand, shows much greater homology with the NBDs of these five ABC proteins, especially in the conserved ATP binding and hydrolysis motifs (Figs. S3 and S4). To understand the contribution of aromatic and negatively-charged residues present in TM helices of DrrB towards drug recognition, a previously published homology model of DrrB 33

was used. This model was developed by the online modeling server Phyre 40, which creates 3-

D models by scanning the generated profile of the query against a fold-library generated from the library of known protein structures and provides models with relatively high accuracy at low sequence identities 40. DrrB contains a total of thirty one aromatic residues and sixteen negatively-charged residues. Of these, twenty four aromatic and four negatively-charged residues are located in the TM helices 35. To determine which residues in DrrB may be important for


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drug recognition, we used the homology model to visualize if a group of aromatic residues may be clustered together in the folded 3-D structure of DrrB thus allowing us to predict which aromatic residues may be part of the drug-binding pocket/cavity. Indeed, based on the strucutre shown in Fig. 1, nine aromatic residues (F65, F69, F177, F179, F185, Y207, F211, F216, , and F269) (shown in red) are seen to form a ‘binding belt’ or ‘ring’ shape in the core of the protein. Four additional aromatic residues (F120, F125, F158, and F252) (also shown in red) are located on the edges of the putative binding cavity.

Fig. 1 Together these thirteen residues were predicted to participate in drug binding and were therefore selected for mutagenesis. The remaining eleven aromatic residues (also located in TM helices) are either located outside of the predicted binding pocket or exposed to the outer surface (shown in green) and were not selected for further analysis in this study. The mutants created in this study were named according to their location and included F65L, F69L, F120L, F125L, F158L, F177L, F179L, F185L, Y207F/S, F211L, F216L, F252L, and F269L. Of the 16 negatively-charged residues found in DrrB, only four are found in the transmembrane helices 35 (Fig. 1A, shown in blue). Because of their location in the TM helices,


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these four residues were also predicted to be critical for drug binding and were selected for mutagenesis. The remaining negatively-charged residues are located in the cytoplasmic loops 35 and were not tested in this study. These four selected residues were mutated to both positive and negatively-charged residues resulting in both conservative and non-conservative mutations, including D57E, D57R, D138E, D138R, E244D, E244R, D254E, and D254R. The location of all mutations created in this study is shown in a cartoon based on the previously reported membrane topology model of the DrrB protein (Fig. 2) 35. Note that the topology model suggested that DrrB contains eight helices with both the N- and the C-termini located in the cytoplasm, however in the homology model shown in Fig. 1, only seven transmembrane helices could be identified. The effect of these twenty two mutations on secondary structure of the proteins and on expression and function of the DrrAB complex was determined, as described below.

Fig. 2 Expression of mutated DrrAB proteins - The expression of wild type and mutated DrrAB proteins was induced as described under Experimental Procedures, and Western blot analysis of


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the membrane fractions was carried out using anti-DrrA or anti-DrrB antibodies. The data in Figs. 3A and 3C show that the expression of DrrA and DrrB was affected to varying degrees in different mutants. The histograms in Figs. 3B and 3D represent average relative expression of DrrA and DrrB determined from three separate experiments. These data show that most of the mutations retained relatively equal DrrA and DrrB expression. However, F179L and D138R mutations showed lower average DrrB expression compared to DrrA, suggesting that DrrA and DrrB interaction may be impaired in these two mutants 32. The D57E mutation showed significantly higher expression of DrrA and DrrB as compared to wild type. Secondary structure analysis - To rule out major alterations in the structure of DrrB mutants, Predictprotein software (predictprotein.org) was used to analyze the secondary structure of each mutant allele. A comparison of mutant structures with wild type DrrB showed no significant change either in the transmembrane helices or in the loop regions of the DrrB protein, thus providing confidence that the mutated DrrB proteins remained largely unchanged. Effect of DrrB mutations on DrrAB-mediated Dox Efflux - A fluorescence-based in vitro assay was used to study Dox efflux in IOVs, as described under Experimental Procedures. Because of the inverted nature of the IOVs, DrrAB-mediated efflux results in accumulation of Dox inside the vesicles, which is seen as quenching of its fluorescence 30. Representative Dox efflux curves obtained with IOVs containing either wildtype DrrAB or DrrAB containing individual aromatic mutations in DrrB are shown in Fig. 4A. The slope of the initial part of the efflux curve (100-200 seconds range) in each case represents their rate of reaction. The relative efficiency of Dox efflux by mutants was determined by dividing the slope of the mutant by slope of wild type. The relative Dox efflux efficiency was then normalized to the expression of DrrB (Fig. 3) in each case by dividing relative slope by relative expression.


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Fig. 3 The normalized relative Dox efflux data from three independent experiments were then used to calculate normalized average relative efficiency and plotted in a histogram shown in Fig. 4B. Of the nine aromatic residues that were seen to form the binding ring (Fig. 1A and 1B), mutations in eight residues (F65L, F69L, F177L, F179L, F185L, Y207F/S, F211L, and F216L) completely inhibited Dox efflux, while F269L retained about 30-40% residual activity (Fig. 4B). Mutations 16 ACS Paragon Plus Environment

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in three (F120L, F158L, F252L) of the remaining four aromatic residues located on the edges showed variable Dox efflux, with F120L showing similar or even higher efflux efficiency than wild type (Fig. 4B). The fourth mutant F125L, however, completely inhibited Dox efflux.

Fig. 4 Effect of mutations in the four negatively-charged residues on Dox efflux was also analyzed, as described above for the aromatic mutations. The efflux efficiency was normalized based on DrrB expression of each mutant (Fig. 3), and the normalized average relative efficiency was calculated and plotted in a histogram (Fig. 5). It was expected that a conservative mutation to another negatively-charged amino acid will not significantly impact Dox efflux function, while change to a positively-charged residue will inhibit activity. However, of the eight mutations tested in this study, six (including both conservative and non-conservative mutations)


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did not exhibit any measurable activity and only two, D57E and E244R, retained partial activity (Figure 5). The non-conservative mutation D57R resulted in a more drastic effect on Dox efflux compared to D57E, as expected. These data suggest that although residue D57 is critical for Dox efflux, substitution to another negatively-charged residue (D57E) still allows partial Dox transport. On the other hand, conservative mutation E244D resulted in complete inhibition of Dox efflux suggesting that this glutamate is absolutely essential for function. Surprisingly, the E244D/R pair of mutations provided unexpected and opposite results so that the nonconservative change to E244R showed high Dox efflux activity (Fig. 5). It is conjectured that the location of the E244 residue at the interface of the membrane and the cytoplasm (determined by topological analysis 35 and also seen in the homology model shown in Fig. 1) may influence its phenotype. Residues at such locations are believed to play a critical role in anchoring the protein to the membrane, and their mutations have been shown to result in anomalous behavior, including structural perturbations 41, 42. Finally, as described in the previous section, of the tested twenty one mutations, F179L and D138R mutants showed relatively low DrrB expression as compared to DrrA, therefore it is unclear if the observed lack of Dox efflux in these two mutants resulted from a direct effect on function of DrrB protein or the impaired DrrA and DrrB interaction. Note that impaired interaction in these mutants may also indicate that these residues are important for function and/or signal transduction between DrrA and DrrB.


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Fig. 5 Effect on multi-drug resistance - Dox efflux analysis described above showed that twelve aromatic and four negatively-charged amino acids found in the TM helices of DrrB are critical for the DrrAB-mediated Dox efflux function. To determine if these mutants were also affected in sensitivity to other MDR drugs, including verapamil, Hoechst33342, and EB, drug sensitivity assays were carried out with selected mutations from each category. The selected aromatic mutants represent different TM helices of DrrB involved in drug binding, and these included F120L (high Dox efflux activity), F158L, F252L, and F269L (low to intermediate activity), and three mutants, F125L, F177L and Y207F, with no activity. Four mutants selected from the negatively-charged residue category included D57R and E244D (two mutants with no activity), D57E (intermediate activity), and E244R (mutant with high activity). To determine the optimum drug concentration range in each case, E. coli cells containing wild type DrrAB or mutant alleles were grown in liquid medium containing a range of drug concentrations. As expected, E. coli cells containing wild type DrrAB (pDX101) showed high resistance to Dox, while control cells containing empty vector (pSU2718) showed the lowest growth at each tested Dox concentration. A representative Dox sensitivity assay is shown in Fig. 6A. Similar assays were optimized for other drugs. For quantitative representation of data and for a comparison of 19 ACS Paragon Plus Environment

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the multidrug resistance profiles of all tested mutants, one drug concentration in the middle of the optimum range was selected for each drug, and relative growth of the mutant (OD600 mutant/OD600 wild type) at that drug concentration was calculated. All assays were repeated at least three times, and the average relative growth was plotted in a histogram (Figs. 6B and 6C). Together, these data show that several DrrB residues critical for Dox binding are also important for binding of verapamil. For example, loss of Dox resistance in D57E, D57R, F125L, F158L, and E244D mutants was accompanied by almost complete loss of verapamil resistance, and high Dox resistance in E244R was accompanied by high verapamil resistance. Phenotype of Y207F, F252L, and F269L mutants, however, suggests that these three residues bind Dox specifically and show minimal overlap with verapamil binding. Of the 11 tested mutations, F120L is the only mutation that gave inconsistent results, exhibiting low Dox resistance but very high Dox efflux (Fig. 4B). Why F120L exhibits opposite Dox efflux and resistance phenotypes is not known; however, this type of anomalous behavior may once again be attributed to its location at the interface of a TM helix and the cytoplasm, as discussed above for the E244R mutation. Interestingly, the data shown in Figs. 6B and 6C also strongly suggest that the Hoechst and EB-binding sites may be independent of the Dox/verapamil binding site. This is evident in particular from the phenotype of E244D mutation, which is extremely Dox and verapamilsensitive but shows very high resistance to Hoechst and EB. On the other hand, D57E, D57R, and F158L mutants exhibited partial loss of Hoechst resistance, and F125L showed complete loss of resistance to Hoechst, thus suggesting that there is also some overlap between Dox/verapamil and the Hoechst binding site. Finally, EB binding also showed variable degree of overlap with Dox, Dox/verapamil and Hoechst binding, as seen in D57E, F158L, F252L, and F269L. However, EB showed specific (exclusive) binding to F120, thus differentiating it from


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the binding of other 3 drugs. Interestingly, mutant F125L retained significant EB resistance in contrast to other 3 drugs, once again separating EB binding from the other drugs. Based on the data shown in Fig. 6 (and Fig. 4B), we conclude that some residues are specific for binding of a particular drug (for example, binding of EB to F120), while other residues show overlapping binding and are shared between multiple drugs. A summary of these data is presented in the working model shown in Fig. 7, and the critical DrrB residues identified in this study are highlighted in the Clustal alignment shown in Fig. S1. We conclude that the aromatic residues indeed play an important role in forming the drug-binding pocket in DrrB, as also seen in other MDR proteins, although the position of the critical aromatic residues in the linear sequence does not appear to be highly conserved between DrrB and Pgp or the Pgp homologs.

Fig. 6


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Fig. 7


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Discussion Multidrug resistance is conferred by specialized membrane proteins that are able to recognize and transport multiple substrates, including compounds with different molecular weights, chemical groups, or topologies 9. Since the discovery of this phenomenon 43-45, scientists have grappled with the question of how a transport protein is able to recognize multiple substrates. The origin and evolution of multidrug resistance in bacteria and the human cancer cells is another important question that remains to be addressed. So far the greatest emphasis on understanding the phenomenon of multidrug resistance has been placed on the mammalian ABC protein Pgp, which confers MDR in cancer cells resulting in loss of effectiveness of chemotherapeutic agents. Indeed an impressive body of information from biochemical and high resolution structural analyses of Pgp has been developed, which together suggests highly malleable binding of substrates to Pgp and provides strong support for the proposed ligandinduced fit mechanism

9, 18, 19

.

In spite of the major strides made with Pgp, however, analysis of bacterial MDR proteins, especially of the ABC superfamily, has lagged behind significantly. To develop a comprehensive understanding of this family, an in-depth analysis of many bacterial MDR systems is needed. In particular, prototype drug resistance systems found in antibiotic producing organisms have the potential to reveal not only if they share multi-specificity with Pgp but also if the molecular mechanism of multi-specificity has remained the same over a large evolutionary timescale. We recently described characterization of a prototype drug resistance system DrrAB found in the producer soil organism S. peucetius. This study showed that contrary to the previously held belief, and in spite of the dedicated nature of this transporter, DrrAB forms a typical multidrug transport system 30, thus making it an excellent model for further investigation


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into the molecular mechanism of poly-specificity in an ancient system. Although DrrB and Pgp as well as other well-studied MDR proteins do not exhibit significant sequence homology (Figs S1 and S2), they may still share a common aromatic residue-based mechanism for poly-specific drug recognition. The aromatic content in TM helices of DrrB is indeed relatively high (15%) and is comparable to the aromatic content of Pgp (18%) 14, 30. Using a previously published homology model of DrrB 33, we predicted that thirteen aromatic amino acids may be critical for drug recognition in this transporter. The data presented in this article indeed support the predictions made from the homology model and confirm that twelve of the predicted thirteen aromatic residues are critical for Dox recognition and efflux. Moreover, residues from several different helices of DrrB (including TM1, TM3, TM4, and TM7) were found to contribute to drug binding. The four tested negatively-charged residues were also found to be critical for Dox efflux and multidrug resistance. Whether these acidic residues are directly involved in recognition of cationic molecules/drugs, as proposed for BmrR and QacR 22-24, or play an indirect role in maintaining structural integrity and conformation of DrrB, will be determined in future studies. The multidrug resistance profile of two acidic mutants shown in Fig. 6C, however, argues against any major change or disruption in conformation of the mutated DrrB proteins. If the structure of these mutant proteins was significantly affected, such a change would be expected to drastically affect resistance to all drugs. This was however not observed - for example, both D57R and E244D mutants lost Dox and verapamil resistance completely but retained much higher resistance to Hoechst and ethidium. Nevertheless, a possible indirect effect of these mutations on resistance cannot be ruled out at this time and will be investigated in detail in the future. It should be pointed out that charged residues are not found in the translocation pathway of the mammalian Pgp 20. However,


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Biochemistry

charged residues are seen in the translocation pathway of C. elegans Pgp as well as bacterial proteins Sav1866 and MsbA 20 although their role in drug binding has so far not been evaluated. A 3-D structure of DrrB will indeed be needed in the future to confirm whether the four acidic residues tested in this study are indeed located inside the translocation pathway of DrrB. Interestingly, multidrug resistance profile of DrrB mutants showed that several tested residues are also involved in binding other MDR substrates, including verapamil, Hoechst and EB, in addition to Dox, thus illuminating the basis for poly-specificity in this transporter. A significant overlap in the binding sites for various drugs was also seen. For example, Dox and verapamil were found to bind many of the same residues, which are separate from the Hoechst and EB binding residues. Overall the presented data show a complex profile and indicate that binding of each drug involves both specific as well as overlapping residues, which may be reflected in their binding affinity at each location. We conjecture that the multiple residues shown to be critical for Dox, Dox/verapamil, Hoechst, and EB recognition in this study are part of a larger (common) drug-binding pocket. Alternatively, they might represent multiple independent binding sites. The former explanation fits the data better, however, as mutations in individual residues (for example, F125L, F185L, and E244D) resulted in complete inhibition of Dox efflux. If they represented independent binding sites, the effect of individual mutations on function would be expected to be much less drastic. Overall this analysis reveals strong similarity between the molecular mechanism of multispecificity in DrrAB and the best understood MDR protein Pgp. In particular, both DrrB and Pgp use aromatic residues for conferring flexibility in drug recognition, and both systems also exhibit specific as well as overlapping drug-binding sites 9, 16, 17, 19, 20, 41, 46, 47. Existence of separate binding sites for Dox and Hoechst in DrrB is also consistent with the existence of R


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(rhodamine and anthracyclines) and H (Hoechst) sites for binding of Dox and Hoechst, respectively, in the common drug-binding pocket of Pgp 9, 48. The proposed ‘substrate-induced fit’ mechanism suggests that a substrate can create its own binding site in a common drugbinding pocket of Pgp by using a combination of residues from different TMs 16, 49. This model was further validated in recent structure-activity analyses 19 with the most recent structural analyses suggesting that creation of flexible drug-binding sites is an intrinsic property of the Pgp 18

. Such flexibility in drug binding is also evident in the drug-binding pocket of DrrB, although

further studies are needed to understand the mechanism by which flexibility is generated in this protein. A working model for the drug-binding pocket of DrrB is presented in Fig. 7. The four panels in this model highlight independent and overlapping DrrB residues shown so far to participate in binding Dox, verapamil, Hoechst and EB. Future studies will focus on further analysis of DrrB mutants to obtain better resolution of the binding sites and to identify residues involved in binding of additional MDR substrates. Another interesting avenue of investigation will be to determine whether mutations in the drug-binding domain of DrrB also impact interaction between previously identified domains of DrrA and DrrB 33, 50, 51 with implications for understanding energy transduction and mechanism of transport in this system. Conflicts of Interest: The authors declare that they have no conflicts of interest with the contents of this article. Supporting information available: Sequence alignments of DrrA and DrrB with other ABC proteins


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Figure Legends Figure 1: Homology model of DrrB. A previously generated homology model of DrrB was analyzed for location of aromatic residues, as described under Experimental Procedures. A, location of the 13 aromatic residues tested in this study (marked in red), remaining 11 aromatic amino acids also located in TM helices but not tested (green), and the four tested negativelycharged residues (blue) is shown. “N” and “C” represent the N-terminal and C-terminal ends of DrrB. The approximate lipid boundary determined by the location of the tryptophan and positively-charged residues 52 in DrrB is shown. B. Ribbon structure of DrrB showing 24 aromatic and four negatively-charged residues located in TM helices. Color coding of residues is the same as seen in Fig. 1A.

Figure 2: A cartoon depiction of the location of various DrrB mutations created in this study. Mutations of aromatic residues and negatively-charged residues are marked; the residues shown in bold represent the aromatic residues. This cartoon is based on the previously published topology model of DrrB 35.

Figure 3: Expression of DrrA and DrrB in wild type and mutants. DrrA and DrrB proteins were analyzed by Western blot analysis, as described under Experimental Procedures. Representative blots are shown in panels A and C. To present the mutants in the order of their amino acid sequence number, the blots were assembled from lanes spliced from different sections of the gel. Each spliced fragment contains its own positive control pDX101 taken from the same gel, which was used for calculation of relative expression of each mutant. The borders between different spliced sections are shown as gaps. The bands were quantified using Multi Gauge V2.3 software


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as described. A, top panel, Western blot analysis of aromatic mutants probed with anti-DrrA antibody; bottom panel, Western blot probed with anti-DrrB antibody. 30 µg of membrane protein was loaded in each well. B, histogram showing average relative expression of DrrA and DrrB in aromatic mutants. The data shown represent average of three separate experiments. C, top panel, Western blot analysis of negatively-charged mutants probed with anti-DrrA antibody; bottom panel, Western blot probed with anti-DrrB antibody. D, histogram showing average relative expression of DrrA and DrrB in negatively-charged mutants. The data shown represent average of three separate experiments.

Figure 4: In vitro analysis of DrrAB-mediated Dox efflux in IOVs containing mutations in aromatic residues. E.coli LE392∆uncIC cells expressing wild type DrrAB or DrrAB containing each aromatic mutation were grown to mid-log phase at 37°C and induced with 0.25 mM IPTG for 3 hours. IOVs were then prepared, as described under Methods. A, in vitro Dox efflux assay was carried out with 250 µM IOVs and 1 µM Dox, as described. Representative efflux curves from one experiment are shown. The higher start point for F269L seen in this particular experiment did not affect its Dox efflux activity and the resulting slope. B, histogram showing efficiency of Dox efflux in IOVs. The average relative efficiency of Dox efflux for each mutant was calculated, as described under Results. The data shown represent average of three separate experiments.

Figure 5: In vitro analysis of DrrAB-mediated Dox efflux in IOVs containing mutations in negatively-charged residues. E.coli LE392∆uncIC cells expressing wild type DrrAB or DrrAB containing each mutation were grown to mid-log phase at 37°C and induced with 0.25 mM IPTG


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for 3 hours. IOVs were then prepared and efflux assay carried out, as described under Experimental Procedures. The average relative efficiency of Dox efflux for each mutant was calculated, as described under Results and plotted in a histogram. The data shown represent average of three independent experiments.

Figure 6: Drug resistance in E. coli cells. E. coli N43 cells expressing wild type DrrAB or DrrAB containing individual mutations were grown at 37°C in liquid M9 medium (or MuellerHinton broth, where indicated) containing a range of drug concentrations (Dox 0-10 µg/mL, Hoechst 0-0.02 µM, Verapamil 0-1000 µM, or EB 0-5 µM) with shaking at 225 rpm for 16-17 hours. Cell growth was measured at 600 nm in a Shimadzu spectrophotometer. A, a typical drug resistance experiment carried out in the presence of Dox using three aromatic mutations. The graph shown is representative of the trends seen. Each experiment was repeated at least three times with similar results seen in each trial. Similar drug resistance experiments were carried out with verapamil, Hoechst 33324, and EB. B, histogram showing summary of results obtained with selected aromatic mutants. One concentration within the optimum range for each drug (Dox- 6 µg/ml; verapamil- 600 µM; Hoechst 33324- 0.1 µM; EB- 2 µM) was selected for quantitative comparisons, as described under Results. The data shown represent average relative growth obtained from multiple experiments carried out with four different drugs. C, histogram showing summary of results obtained with selected negatively-charged mutants. The data were analyzed as described in B above, and the shown data represent average relative growth obtained from multiple experiments carried out with four different drugs.


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Biochemistry

Fig. 7: A model for the predicted drug-binding cavity of DrrB seen from the cytoplasmic side. This working model combines the homology model shown in Fig. 1 with the data obtained from analysis of mutants in aromatic and negatively-charged residues presented in this article. The transmembrane helices are labeled 1-7. Mutations resulting in less than 40% Dox efflux (Top Panel) or H33342, EB, and verapamil resistance (Bottom panels) are shown as magenta. Mutations that retained between 40-60% of Dox efflux or drug resistance are shown in gray color in each panel, and residues that retained greater than 60% Dox efflux or drug resistance are not shown. Top Panel, mutations in13 aromatic and four negatively-charged residues were tested for their effect on Dox efflux. All of the mutations, except F269L and F120L, showed a drastic effect and are shown in magenta color. F269 is shown in gray, while F120 is not shown as the F120L mutation retained wild-type level of Dox efflux. Bottom panels, a selected group, consisting of 11 mutants (described under Results), was tested for the effect of mutations on their multi-drug resistance profile. Bottom left panel, resistance to H 33342. Of the tested mutants, only residue F125 was found to be critical for resistance to H33342, which is shown in magenta color. Three residues (F158, F177, and Y207) conferred only partial loss of H 33342 resistance and are shown in gray color; Bottom center panel, resistance to EB. Of the tested mutations, D57, F120, F252, and F269 were found to be critical for EB resistance and are marked in magenta color. Residues F125, F158, F177, and Y207 affected EB resistance only partially and are shown in gray; Bottom right panel, resistance to verapamil. Mutations in residues F125, F158, F177, D57, and E244 affected verapamil resistance drastically and are shown in magenta color. Mutation in F252 affected verapamil resistance partially and is shown in gray color.


Biochemistry

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Role of Aromatic and Negatively Charged ... - ACS Publications

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