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H+ antiport: complete cysteine-scanning mutagenesis and the protein engineering approach
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Mechanisms of drug/HR antiport: complete cysteine-scanning mutagenesis and the protein engineering approach Norihisa Tamura, Satoko Konishi and Akihito Yamaguchi The notorious difficulty of elucidating structures of membrane transporters by crystallography has long prevented our understanding of active transport mechanism coupled with ion/ proton transport. The determination of the first crystal structure of the drug/Hþ antiporter AcrB was a breakthrough for structurebased understanding of drug/Hþ antiport. However, although AcrB is a major multidrug exporter in Gram-negative organisms, the majority of bacterial drug exporters are major facilitator superfamily (MFS) drug transporters. As no crystal structures have been solved for MFS transporters, the alternative proteinengineering methods are still very useful for estimating structures and functions of drug/Hþ antiporters. This review describes this alternative approach for investigating the structure and function of tetracycline/Hþ antiporters. Addresses Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan e-mail: [email protected]
Current Opinion in Chemical Biology 2003, 7:570–579 This review comes from a themed issue on Mechanisms Edited by Shahriar Mobashery and John P Richard 1367-5931/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2003.08.014
Abbreviations AMS 4-acetamido-40 -meleimidylstilbene-2,20 -disulfonic acid MFS major facilitator superfamily NEM N-ethyl maleimide
Introduction The mechanism of bacterial tetracycline resistance is unique, mainly involving active drug efflux [1]. Until a rush of the findings of multidrug exporters in the 1990s [2], tetracycline was, excepting antiseptics, almost the only antibiotic that suffered efflux-based resistance [3]. Although it was known in the 1960s that tetracycline resistance is based on the decreased accumulation of tetracycline in bacterial cells [4], the tetracycline exporter was not identified until 1980 [1]. This was the first identified antibiotic efflux transporter, a metal-tetracycline/Hþ antiporter [5], which belongs to a major facilitator superfamily (MFS) [6]. Since then, many homologues of tetracycline exporters have been found and classified into class A–H, and K–L [7]. Most are encoded in R-plasmids. Current Opinion in Chemical Biology 2003, 7:570–579
Although some catalyze Naþ and Kþ exchange with Hþ [8], no physiological substrate has been found. The structure and function of tetracycline/Hþ antiporter has been thoroughly studied and thus it has become almost the paradigm for molecular biological studies of drug/Hþ antiport mechanisms. The hydrophobic and metastable nature of membrane transport proteins has long prohibited the crystal structure determination, until a recent breakthrough with ABC transporters [9,10] and a multidrug efflux transporter [11]. Very recently, Kaback et al. and Lemieux et al. solved the crystal structures of MFS-type secondary transporters in Escherichia coli — lac permease and glycerol-3-phosphate transporter, respectively (personal communication). However, the situation is still unaltered in many other transporters, including drug exporters. In such cases, site-directed mutagenesis and chemical modification studies are useful alternatives to investigate the structure and mechanism of membrane transport [12,13–15]. Recent crystallographic analysis has corroborated the structure of AcrB, which was estimated by site-directed chemical modification [11]. As cysteine-scanning mutagenesis is especially useful for structural and functional studies on membrane transporters, many have been studied using this technique, including lac permease [16], P-glycoprotein [17,18], glucose transporter [19,20], glutamate transporter [21,22], prostaglandin transporter [23] and mitochondrial oxoglutarate transporter [24]. With respect to drug exporters, complete cysteine-scanning mutants of 400 amino acid residues of tetracycline/Hþ antiporter TetA(B) were constructed and extensively analyzed [25]. Here we briefly review the structural and functional implications of the studies on the complete cysteine-scanning mutants of TetA(B).
Metal-tetracycline/HR antiport Tetracycline enters cells by simple diffusion through the lipid bilayer region of the plasma membrane as a protonated neutral form (Figure 1) [26]. Then, in the cell interior, it loses a proton and chelates with Mg2þ. The resulting monovalent cation is exported by TetA(B) coupled with proton influx (Figure 1) [5]. Thus TetA(B) functions as a metal–tetracycline/Hþ antiporter.
Membrane topology Although crystal structures of membrane proteins are notoriously elusive [27], the transmembrane structures themselves are rather simple, that is, they mostly comprise www.current-opinion.com
Mechanisms of drug/HR antiport Tamura, Konishi and Yamaguchi 571
Figure 1
Figure 2
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Tetracycline transport cycle in resistant E. coli cells. (a) Chemical structure of tetracycline. W1–4 represent water molecules. (b) Tetracycline influx and TetA(B)-mediated efflux in E. coli. TH2, TH and THMgþ indicate a neutral form, a monoanionic form, and a Mg2þ chelate complex of tetracycline, respectively.
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a-helix bundle [28]. Therefore, if we can determine the numbers and the ranges of the transmembrane segments and their arrangement in the membrane plane, we can roughly estimate the membrane protein structure. The first step is to determine the membrane-spanning topology. The most popular method is by reporter-enzyme fusion [29,30]. However, it is questionable whether the fusion proteins reflect their native structures. We have developed an alternative method based on the site-directed competitive chemical modification of cysteine-introducing mutants into a cysteine-free derivative of a membrane protein [31] (Figure 2a,b). In this method, two kinds of maleimide derivative, membrane-permeable N-ethyl maleimide (NEM) and membrane-impermeable 4-acetamido40 -meleimidylstilbene-2,20 -disulfonic acid (AMS), are used. The inside/outside localization of introduced www.current-opinion.com
Determination of the topology of tetracycline/Hþ antiporter TetA(B) by site-directed competitive chemical modification of Cys mutants. (a) Principle of the determination of the inside/outside/embedded location of cysteine residues. (b) An example of [14 C] NEM binding to Cys mutants introduced to the periplasmic, cytoplasmic and membrane regions and the competitive inhibition with AMS pretreatment. Labeling was performed in intact cells followed by preparation of the membrane. Radioactive bands were detected by autoradiography of the SDS electrophoresis gel. (c) Topology determined by the competitive labeling. Open and closed ovals indicate the Cys-introduced positions of which NEM binding was not affected and prevented by AMS pretreatment, respectively.
cysteine residues is judged from competitive binding of these two compounds in intact E. coli cells expressing Cys mutants. This method is quite reliable as Cys mutants retaining the drug transport activity can be used for the Current Opinion in Chemical Biology 2003, 7:570–579
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Figure 3
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experiment. The results are shown in Figure 2c, indicating that TetA(B) has 12 transmembrane segments [31]. The same method has been applied to topology determination of a multidrug transporter AcrB [32] and a macrolidespecific ABC-type exporter MacB [33]. The exactness of the estimation by this method was later supported by X-ray crystal structure determination of AcrB [11].
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If such a water-filled channel exists at the center of the TetA(B) molecule, why doesn’t TetA(B) cause uncoupling of the membrane? To answer this question, we investigated the accessibility of membrane-impermeable maleimide AMS to the NEM-reactive Cys mutants in the transmembrane region. For example, in the case of TM5, AMS prevented NEM binding to G150C, G149C, P146C and G145C but does not affect the binding to L142C,
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Therefore, we replaced all 400 amino acid residues of TetA(B), except for the first methionine, with cysteine one by one [25]. It was revealed that TM5, TM8 and TM11 face a water-filled channel similar to TM2. For TM1, TM4, TM7 and TM10, the N-terminal halves are embedded in the membrane and the C-terminal halves face the water channel (Figure 4a). Because oddnumbered helices penetrate the membrane in the N to C direction while even-numbered ones penetrate in the C to N direction TM1 and TM7 face the water channel at their periplasmic halves, and TM4 and TM10 face it at their cytoplasmic halves. Thus, the channel wall is composed of TM1, TM2, TM5, TM7, TM8 and TM11 in the periplasmic half and TM2, TM4, TM5, TM8, TM10 and TM11 in the cytoplasmic half (Figure 4b).
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However, with TM2, the Cys-scanning mutants did not show the continuous NEM-non-reactive region (Figure 3b). Instead, the transmembrane region showed periodical appearance of NEM-reactive and non-reactive positions. The NEM-reactive positions are located on the same side of the a-helix, indicating that TM2 faces the water-filled channel (Figure 3c). This suggests that we can distinguish the water-channel-facing helices and the membrane-embedded helices.
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Because the reactivity of a maleimide compound with a sulfhydryl group is very low in the hydrophobic environment, it is possible to determine the exact range of a membrane-spanning region using NEM reactivity with Cys mutants. Figure 3a shows the typical [14 C] NEMlabeling results of cysteine-scanning mutants around transmembrane segment 3 (TM3) [34]. A 20 residuelong continuously NEM non-reactive region was observed in TM3, indicating the membrane-embedded transmembrane a-helix. The same continuously NEMnon-reactive regions were also observed in TM6 [35], TM9 [36] and TM12 [25]. A similar observation was also reported for EmrE [37].
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[14 C] NEM labeling of the cysteine-scanning mutants of TetA(B). (a) Labeling around TM3 and its flanking regions. The underline indicates the predicted TM3 region. (b) Labeling around TM2 and its flanking regions. The underline indicates the predicted TM2 region. (c) 3D model of predicted TM2. Closed ovals with outlined letters indicate the NEM reactive positions. White ovals indicate the NEM nonreactive positions. The gray arrow is a predicted water-filled channel.
G139C and F138C in intact cells (Figure 4a), while AMS prevents NEM binding of all these Cys mutants in sonicated membrane fragments [25]. These observations www.current-opinion.com
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Figure 4
Results of competitive labeling of the complete cysteine-scanning mutants of TetA(B) with maleimide compounds. (a) Determined topology of TetA(B). Blue and red letters indicate the NEM-reactive and non-reactive positions, respectively. Pink and sky blue regions of transmembrane regions indicate the predicted membrane-embedded and water-exposed regions, respectively. Yellow and white circles indicate the positions at which NEM binding is prevented and not affected, respectively, by AMS pretreatment in intact cells. Green horizontal lines indicate the predicted permeability barrier. (b) Predicted arrangements of transmembrane a-helix wheels at cytoplasmic and periplasmic sides. In each wheel, amino acid residues only in the cytoplasmic or periplasmic half of the transmembrane helix are depicted. Amino acid residues on purple and white backgrounds indicate the NEM reactive and non-reactive positions, respectively.
indicate that the permeability barrier against AMS molecules exists at the middle of TM5. Because AMS can access all of the ‘hidden Cys residues’ in sonicated membrane fragments, the permeability barrier is very thin with the thickness of about one a-helical turn. www.current-opinion.com
Similar barriers were also observed in all channel-facing helices. In addition, the positions of barriers in all helices were located at almost the same membrane depth except for TM1 (Figure 4a). In TM1, the barrier position is near the periplasmic end, suggesting that the correct TM1 Current Opinion in Chemical Biology 2003, 7:570–579
574 Mechanisms
position might be shifted toward the cytoplasmic direction. The AMS competition experiment clearly indicates the presence of the permeability barrier having one a-helical turn thickness at the middle of the waterfilled channel. This permeability barrier keeps TetA(B) from uncoupling the membrane.
Next, we constructed all combinations of double Cys mutations at the cytoplasmic and periplasmic ends of the transmembrane segments. Figure 5a shows the disulfide linkages on the topology model. Figure 5b,c show the putative helix arrangements at the cytoplasmic and periplasmic surface, respectively, with disulfide linkages. At the cytoplasmic side, TM4 formed disulfide linkages with TM8, TM10 and TM11. TM10 formed linkages with TM4 and TM2. However, at the periplasmic side, TM4 and TM10 did not form disulfide linkages with any other helices. Instead, TM1 and TM7 formed disulfide linkages with each other and with other helices at the periplasmic side. Conversely, TM2, TM8 and TM11 formed disulfide linkages at both sides. These observations indicate that the channel-facing helices are changed between periplamic and cytoplasmic sides. TM4 and 10 face the water channel at the cytoplasmic side, whereas
Helix packing To determine the helix arrangement in the membrane plane, we adopted a method based on the detection of the intramolecular disulfide linkage of double Cys mutants. The formation of a disulfide linkage was detected by the band shift on SDS polyacrylamide gel electrophoresis after oxidation with Cu2þ/o-phenanthroline. It was revealed that the helix bundle of TetA(B) is a circular form, that is, the first and last helices are close to each other [38].
Figure 5
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Disulfide cross-linking results. (a) Disulfide cross-linking is shown on the topology of TetA(B). Cysteine-introduced positions are depicted by bold letters. Disulfide cross linking on the cytoplasmic and periplasmic sides are depicted by red and green lines, respectively. (b) Helix arrangement of TetA(B) at cytoplasmic side based on cross-linking results. (c) Helix arrangement at periplasmic side. (d) Helix arrangement at periplasmic side when tetracycline binds to TetA(B). Red lines indicate the disulfide cross-linking. Red broken lines indicate the disulfide cross-linking disappeared in the tetracycline-binding form. Current Opinion in Chemical Biology 2003, 7:570–579
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TM1 and 7 face at the periplasmic side. These observations coincide with the results of NEM accessibility (Figure 4b). Similar helix arrangement and the exchange of the water-channel-facing transmembrane helices are also observed in the oxalate transporter using electron microscopy analysis of two-dimensional crystals [39].
affect the transport activity of the wild-type TetA(B), the S65C mutant is completely inactivated by NEM [43]. Among 36 cysteine-scanning mutants between K63C and Y98C, there are only two Cys mutants, S65C and L97C, that are inactivated by NEM [44]. Interestingly, these hot spots are symmetrically located across TM3.
Significant alteration in the cross-linking pattern was observed when tetracycline bound to TetA(B). As shown in Figure 5, in the presence of tetracycline, TM1 did not form a cross-link with TM7 or TM11 in the periplasmic side. TM2 also did not form a cross-link with TM7, whereas cross-links with TM8, TM11 and TM12 were unaltered. These observations suggest that substrate binding might cause TM1 to move away from TM7/11, resulting in the enlargement of the periplasmic opening of the channel.
Surprisingly, the only functionally essential residues in this motif are Gly62 and Arg70, in addition to Asp66. The mutants of Arg67, Gly69 and Arg71 retain the transport activity [45]. In addition, triple mutants (K63L/R67L/ R71L) also retain the transport activity, indicating that the polycationic nature of this loop is not necessary for the function. The general notion that conserved residues are functionally important might have to be changed.
Possible gate for substrate entering into the exporter Tetracycline/Hþ antiporters have a conserved sequence motif, 62 GXXXDRXGRR71 , in cytoplasmic loop2–3 between TM2 and TM3 (Figure 6), which is also common to MFS transporters [40]. Although TetA(B) has seven conserved acidic residues in the hydrophilic loop region, Asp66 in this motif is the only essential residue of which mutations to any other amino acid residues result in complete loss of transport function [41]. Ser65 is not conserved and its mutation to cysteine did not affect the function; however, position 65 is a hot spot for sulfhydryl inactivation [42]. Although NEM does not
With respect to Arg70, R70K retains some activity but any other mutants are inactive; however Hg2þ or Co2þ can restore the transport activity of R70C via mercaptide formation [46]. These observations indicate that positive charge at position 70 is essential. This positive charge works via ion pairing with a negative charge of Asp120 located in the next loop between TM4 and TM5 [47]. D120N mutation partially restores the activity of the R70A mutant, while D66A mutation has no such effect [47]. The charge exchange mutant R70D/D120R also shows transport activity, although R70D has no activity. The R70C/D120C double mutant also retained activity slightly lower than that of the D120C single mutant with activity significantly higher than that of the R70C single mutant. However, the R70C/D120C double mutant is
Figure 6
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Functionally essential or important residues described in the text are shown on the TetA(B) topology. Pink and blue ovals indicate acidic and basic residues, respectively. White rectangles indicate hot spots for inactivation of Cys mutants with NEM. Red and black rectangles indicate the first mutation sites resulting in loss of resistance and the second mutation sites that suppress the effect of the first mutation, respectively. The dotted line indicates the ion pair. The MFS motif is shown on loop2–3. www.current-opinion.com
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Figure 7
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Postulated model for tetracycline/Hþ antiport mediated by TetA(B). In this model, TM6, 10, 11 and 12 are removed for easy inspection. Metaltetracycline complex enters into TetA(B) through the gate composed of loop2–3 and loop4–5. Then it is transported through the water-filled channel, probably close to the wall composed of helices 1, 2 and 4. Finally, it is released from TetA(B) near Arg101. Helix 3 is embedded in the membrane, while Asp84 may be exposed by substrate-induced conformational change during transport function. The proton translocation pathway might be separated from the substrate pathway and located in the hydrophobic core composed of helices 7, 8 and 9.
completely inactivated with Hg2þ, probably because the side chains are bridged with Hg2þ, indicating that the dynamic association/dissociation of the Arg70–Asp120 ion pair is required for tetracycline transport function. These observations suggest that loop2–3 may function as a gate for tetracycline entry into TetA(B) (Figure 7).
Functional charged residues in the transmembrane region In the transmembrane region of TetA(B), there are three acidic residues (Asp15, 84 and 285) and two basic residues (Arg101 and His257). These five charged residues are highly conserved in tetracycline exporters [48–50]. Sitedirected mutagenesis studies revealed that all of them are functionally important [48,49,51] (Figure 6). Out of three acidic residues, Asp285 is essential for TetA(B) function. When it is replaced with any other residues, the resulting mutant lost not only tetracycline transport activity but also the ability for tetracycline binding [52]. Asp285 is located at the middle of TM9, a fully membrane-embedded transmembrane a-helix [36]. His257 is located at the middle of TM8 and plays an important role in proton Current Opinion in Chemical Biology 2003, 7:570–579
translocation [50] probably via ion pairing with Asp285. We found a fascinating second-site suppressor mutant from D285N mutant [53]. In that mutant, Ala220 was replaced with Glu. The resulting mutant recovered almost full drug resistance and about half of tetracycline export activity. Ala220 is located in TM7 at almost the same membrane depth as His257 and Asp285. These observations might implicate that the triplet of TM7, TM8 and TM9 forms a proton translocation pathway (Figure 7). Arg101 is another essential charged residue in the transmembrane region and is located near the periplasmic end of TM4 [49]. Considering the symmetry of sulfhydryl hot spots, S65C in loop2–3 and L97C in loop3–4, Arg101 may play a similar role to Arg70 in loop2–3, as an exit gate for substrate translocation (Figure 7).
Second-site suppressor mutations indicate structural and functional coupling between separated residues TetA(B) gave various second-site suppressor mutations other than D285N/A220E, as described above. The www.current-opinion.com
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typical one is the L30S mutation, which recovers drug resistance from the G62L mutant (Figure 6) [54]. Gly62 is located in cytoplasmic loop2–3 and is the first residue of the MFS conserved motif [40]. Leu30 is located at the opposite side in loop1–2. Interestingly, when NEM accessibility to L29C is used for a measure of the local conformation around loop1–2, position 29 is exposed to [14 C] NEM in the wild-type TetA(B) having only the L29C mutation, which is neutral for the transport function. However, position 29 is hidden in G62L mutant, indicating that the effect of the Gly62Leu mutation in cytoplasmic loop2–3 is transmitted to the periplasmic loop1–2 to hide position 29. However, the second-site Leu30Ser mutation recovers the effect of G62L and exposes position 29 to the medium. Such remote conformational linkage is the cause for recovery in drug resistance. Similar transmembrane conformational linkage is also observed across TM11. That is, the loss of activity with Gly332Ser mutation in loop10–11 is recovered by Ala354Asp mutation in loop11–12 (Figure 6) [55]. This is also due to the occlusion of position 354 with G332S mutation and exposition again with A354D second-site mutation [55]. Interestingly, G332S mutation is also suppressed by L30S mutation and G62L mutation is suppressed by A354D mutation, indicating that TM2 and TM11 are close to each other and conformationally linked [55], which is supported by the helix arrangement estimated by disulfide cross-linking (Figure 5).
results suggest that proton translocation and substrate transport are separated and only conformationally linked. This is consistent with the recent crystallographic study of lac permease, in which the substrate pathway and proton translocation pathway are separated by at least 6 A˚ . RND-type multidrug exporter AcrB is also driven by remote-conformational coupling [11]. Therefore, the remote-conformational coupling between proton translocation and the substrate transport may be a general rule in a secondary transporter.
Conclusion Site-directed mutagenesis and the chemical modification studies on tetracycline/Hþ antiporter TetA(B) as a model for drug/Hþ antiport revealed the presence of a central water-filled channel with a thin permeability barrier at the middle of the channel. As the distances between transmembrane helices across the channel are increased at the periplasmic side when tetracycline binds to TetA(B), the channel acts as at least a part of the tetracycline translocation pathway. However, some residues important or essential for substrate translocation are located in the membrane-embedded face of the helices (Figure 7), suggesting the conformational change and substrate-induced exposure of these residues. Our results indicate that tetracycline enters TetA(B) through a gate between loop2–3 and loop4–5. It is then transported through the water-filled channel, probably close to the wall composed of N-terminal helices such as TM1, TM2 and TM4. Finally it is released from an exit gate around Arg101. TM3 is embedded in the membrane, while Asp84 may be exposed by substrate-induced conformational change during transport function. The residues important for proton translocation are mainly located on the membrane-embedded surface of the C-terminal helices. The proton translocation pathway seems to be separated from the water-filled channel and the substrate translocation pathway (Figure 7). Our www.current-opinion.com
Acknowledgements We thank Tomomi Kimura-Someya, Yuichi Someya, and past and present members of our laboratory.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Nikaido H: Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 1994, 264:382-388.
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Rouch DA, Cram DS, DiBerardino D, Littlejohn TG, Skurray RA: Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracyclineand sugar-transport proteins. Mol Microbiol 1990, 4:2051-2062.
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Izaki K, Arima K: Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli. J Bacteriol 1965, 91:628-633.
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Yamaguchi A, Udagawa T, Sawai T: Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein. J Biol Chem 1990, 265:4809-4813.
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Marger MD, Saier MH: A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci 1993, 18:13-20.
7.
Hansen LM, McMurry L, Levy SB, Hirsh DC: A new tetracycline resistance determinant, TetH, from Pasteurella multocida specifying active drug efflux of tetracycline. Antimicrob Agents Chemother 1993, 37:2699-2705.
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Krulwich TA, Jin J, Guffanti AA, Bechhofer H: Functions of tetracycline efflux proteins that do not involve tetracycline. J Mol Microbiol Biotechnol 2001, 3:237-246.
9.
Chang G, Roth CB: Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 2001, 293:1793-1800. First crystal structure of ABC protein. 10. Locher KP, Lee AT, Rees DC: The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 2002, 296:1091-1098. Crystal structure of ABC transporter. 11. Murakami S, Nakashima R, Yamashita E, Yamaguchi A: Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 2002, 419:587-593. First crystal structure determination of multidrug/Hþ antiporter. 12. Kaback HR, Sahin-Toth M, Weinglass AB: The kamikaze approach to membrane transport. Nat Rev Mol Cell Biol 2001, 2:610-620. Approach for structure and function of a transporter without a crystal structure. Current Opinion in Chemical Biology 2003, 7:570–579
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13. Loo TW, Bartlet MC, Clarke DM: Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein: direct evidence for the substrateinduced fit mechanism for drug binding. J Biol Chem 2003, 278:13603-13606. The authors investigated the substrate-induced fit conformational change in the transmembrane segments of P-glycoprotein with oxidative crosslinking of double cysteine mutants. 14. Loo TW, Clarke DM: Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil. J Biol Chem 2001, 276:14972-14979. The drug binding site of P-glycoprotein was determined in the cysteinescanning mutants with thiol-reactive analog of P-gp stimulator MTSverapamil. 15. Ding PZ, Weissborn AC, Wilson TH: Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli. J Membr Biol 2001, 183:33-38. Cysteine-scanning mutants of the melibiose carrier in E. coli were constructed and the reactivity with hydrophilic sulfhydryl reagent PCMBS was measured. The authors concluded that helix VI is imbedded in phospholipid and does not face the aqueous channel. 16. Frillingos S, Sahin-Toth M, Wu J, Kaback HR: Cys-scanning mutagenesis: a novel approach to structure-function relationships in polytopic membrane proteins. FASEB J 1998, 12:1281-1299. 17. Loo TW, Clarke DM: Determining the structure and mechanism of the human multidrug resistance P-glycoprotein using cysteine-scanning mutagenesis and thiol modification techniques. Biochim Biophys Acta 1999, 1461:315-325. 18. Blott EJ, Higgins CF, Linton KJ: Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein. EMBO J 1999, 18:6800-6808. 19. Hruzz PW, Mueckler MM: Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter. Biochemistry 2000, 39:9367-9372. 20. Lo B, Silverman M: Cysteine scanning mutagenesis of the segment between putative transmembrane helices IV and V of the high affinity NaR/glucose cotransporter SGLT1: evidence that this region participates in the NaR and voltage dependence of the transporter. J Biol Chem 1998, 273:29341-29351. 21. Slotboom DJ, Konings WN, Lolkema JS: Cysteine-scanning mutagenesis reveals a highly amphipathic, pore-lining membrane-spanning helix in the glutamate transporter GltT. J Biol Chem 2001, 276:10775-10781. 22. Grunewald M, Menaker D, Kanner BI: Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1. J Biol Chem 2002, 277:26074-26080. 23. Chan BS, Satriano JA, Schuster VL: Mapping the substrate binding site of the prostaglandin transporter PGT by cysteine scanning mutagenesis. J Biol Chem 1999, 274:25564-25570. 24. Stipani V, Cappello AR, Daddabbo L, Natuzzi D, Miniero DV, Stipani I, Palmieri F: The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents. Biochemistry 2001, 40:15805-15810. 25. Tamura N, Konishi S, Iwaki S, Kimura-Someya T, Nada S, Yamaguchi A: Complete cysteine-scanning mutagenesis and site-directed chemical modification of the TN10-encoded metal-tetracycline/HR antiporter. J Biol Chem 2001, 276:20330-20339. Most detailed analysis of whole complete cysteine-scanning mutants of TetA(B). 26. Yamaguchi A, Ohmori H, Kaneko-Ohdera M, Nomura T, Sawai T: Delta pH-dependent accumulation of tetracycline in Escherichia coli. Antimicrob Agents Chemother 1991, 35:53-56. 27. Caffrey M: Membrane protein crystallization. J Struct Biol 2003, 142:108-132. Current Opinion in Chemical Biology 2003, 7:570–579
28. Kim S, Cross TA: Uniformity, ideality, and hydrogen bonds in transmembrane alpha-helix. Biophys J 2002, 83:2084-2095. 29. Boyd D, Beckwith M: Determinants of membrane protein topology. Proc Natl Acad Sci USA 1987, 84:8525-8529. 30. Gott P, Boos W: The transmembrane topology of the snglycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions. Mol Microbiol 1988, 2:655-663. 31. Kimura T, Ohnuma M, Sawai T, Yamaguchi A: Membrane topology of the transposon 10-encoded metal-tetracycline/HR antiporter as studied by site-directed chemical labeling. J Biol Chem 1997, 272:580-585. 32. Fujihira E, Tamura N, Yamaguchi A: Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli. J Biochem (Tokyo) 2002, 131:145-151. 33. Kobayashi N, Nishino K, Hirata T, Yamaguchi A: Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli. FEBS Lett 2003, 546:241-246. 34. Kimura T, Shiina Y, Sawai T, Yamaguchi A: Cysteine-scanning mutagenesis around transmembrane segment III of Tn10encoded metal-tetracycline/HR antiporter. J Biol Chem 1998, 273:5243-5247. 35. Konishi S, Iwaki S, Kimura-Someya T, Yamaguchi A: Cysteinescanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/HR antiporter. FEBS Lett 1999, 461:315-318. 36. Kimura T, Suzuki M, Sawai T, Yamaguchi A: Determination of a transmembrane segment using cysteine-scanning mutants of transposon Tn10-encoded metal-tetracycline/HR antiporter. Biochemistry 1996, 35:15896-15899. 37. Mordoch SS, Granot D, Lebendiker M, Schuldiner S: Scanning cysteine accessibility of EmrE, an HR-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes. J Biol Chem 1999, 274:19480-19486. 38. Kubo Y, Konishi S, Kawabe T, Nada S, Yamaguchi A: Proximity of periplasmic loops in the metal-tetracycline/HR antiporter of Escherichia coli observed on site-directed chemical crosslinking. J Biol Chem 2000, 275:5270-5274. 39. Hirai T, Heymann JAW, Shi D, Sarkar R, Maloney PC, Subramaniam S: Three-dimensional structure of a bacterial oxalate transporter. Nat Struct Biol 2002, 9:597-600. Helix arrangement of a secondary transporter determined by electron microscopic analysis of two-dimensional crystal. 40. Maiden MCJ, Davis EO, Baldwin SA, Moore DCM, Henderson PJF: Mammalian and bacterial sugar transport proteins are homologous. Nature 1987, 325:641-643. 41. Yamaguchi A, Nakatani M, Sawai T: Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/HR antiporter encoded by transposon Tn10 of Escherichia coli. Biochemistry 1992, 31:8344-8348. 42. Yamaguchi A, Kimura T, Sawai T: Hot spots for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motifs of the metal-tetracycline/HR antiporter in Escherichia coli. J Biochem 1994, 115:958-964. 43. Yamaguchi A, Ono N, Akasaka T, Noumi T, Sawai T: Metaltetracycline/HR antiporter of Escherichia coli encoded by a transposon Tn10: the role of the conserved dipeptide, Ser65Asp66, in tetracycline transport. J Biol Chem 1990, 265:15525-15530. 44. Kimura T, Shiina Y, Sawai T, Yamaguchi A: Cysteine-scanning mutagenesis around transmembrane segment III of Tn10encoded metal-tetracycline/HR antiporter. J Biol Chem 1998, 273:5243-5247. 45. Yamaguchi A, Someya Y, Sawai T: Metal-tetracycline/HR antiporter of Escherichia coli encoded by a transposon Tn10: The role of a conserved sequence motif, GXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3. J Biol Chem 1992, 267:19155-19162. www.current-opinion.com
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46. Someya Y, Yamaguchi A: Mercaptide formed between the residue Cys70 and Hg2R or Co2R behaves as a functional positively charged side chain operative in the Arg70!Cys mutant of the metal-tetracycline/HR antiporter of Escherichia coli. Biochemistry 1996, 35:9385-9391.
51. Yamaguchi A, Samejima T, Kimura T, Sawai T: His257 is a uniquely important histidine residue for tetracycline/HR antiport function but not mandatory for full activity of the transposon Tn10-encoded metal-tetracycline/HR antiporter. Biochemistry 1996, 35:4359-4364.
47. Someya Y, Kimura-Someya T, Yamaguchi A: Role of the charge interaction between Arg70 and Asp120 in the Tn10-encoded metal-tetracycline/HR antiporter of Escherichia coli. J Biol Chem 2000, 275:210-214.
52. Kimura T, Yamaguchi A: Asp-285 of the metal-tetracycline/HR antiporter of Escherichia coli is essential for substrate binding. FEBS Lett 1996, 388:50-52.
48. Yamaguchi A, Akasaka T, Ono N, Someya Y, Nakatani M, Sawai T: Metal-tetracycline/HR antiporter of Escherichia coli encoded by a transposon Tn10: roles of aspartyl residues located in the putative transmembrane helices. J Biol Chem 1992, 267:7490-7498.
53. Yamaguchi A, O’yauchi R, Someya Y, Akasaka T, Sawai T: Second-site mutation of Ala220 to Glu or Asp suppresses the mutation of Asp285 to Asn in the transposon Tn10-encoded metal-tetracycline/HR antiporter of Escherichia coli. J Biol Chem 1993, 268:26990-26995.
49. Kimura T, Nakatani M, Kawabe T, Yamaguchi A: Roles of conserved arginine residues in the metal-tetracycline/HR antiporter of Escherichia coli. Biochemistry 1998, 37:5475-5480.
54. Kimura T, Sawai T, Yamaguchi A: Remote conformational effects of the Gly-62!Leu mutation of the Tn10-encoded metaltetracycline/HR antiporter of Escherichia coli and its secondsite suppressor mutation. Biochemistry 1997, 36:6941-6946.
50. Yamaguchi A, Adachi K, Akasaka T, Ono N, Sawai T: Metaltetracycline/HR antiporter of Escherichia coli encoded by a transposon Tn10: Histidine 257 plays an essential role in HR translocation. J Biol Chem 1991, 266:6045-6051.
55. Kawabe T, Yamaguchi A: Transmembrane remote conformational suppression of the Gly-332 mutation of the Tn10-encoded metal-tetracycline/HR antiporter. FEBS Lett 1999, 457:169-173.
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Current Opinion in Chemical Biology 2003, 7:570–579
Mechanisms of drug/HR antiport: complete cysteine-scanning mutagenesis and the protein engineering approach Norihisa Tamura, Satoko Konishi and Akihito Yamaguchi The notorious difficulty of elucidating structures of membrane transporters by crystallography has long prevented our understanding of active transport mechanism coupled with ion/ proton transport. The determination of the first crystal structure of the drug/Hþ antiporter AcrB was a breakthrough for structurebased understanding of drug/Hþ antiport. However, although AcrB is a major multidrug exporter in Gram-negative organisms, the majority of bacterial drug exporters are major facilitator superfamily (MFS) drug transporters. As no crystal structures have been solved for MFS transporters, the alternative proteinengineering methods are still very useful for estimating structures and functions of drug/Hþ antiporters. This review describes this alternative approach for investigating the structure and function of tetracycline/Hþ antiporters. Addresses Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan e-mail: [email protected]
Current Opinion in Chemical Biology 2003, 7:570–579 This review comes from a themed issue on Mechanisms Edited by Shahriar Mobashery and John P Richard 1367-5931/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2003.08.014
Abbreviations AMS 4-acetamido-40 -meleimidylstilbene-2,20 -disulfonic acid MFS major facilitator superfamily NEM N-ethyl maleimide
Introduction The mechanism of bacterial tetracycline resistance is unique, mainly involving active drug efflux [1]. Until a rush of the findings of multidrug exporters in the 1990s [2], tetracycline was, excepting antiseptics, almost the only antibiotic that suffered efflux-based resistance [3]. Although it was known in the 1960s that tetracycline resistance is based on the decreased accumulation of tetracycline in bacterial cells [4], the tetracycline exporter was not identified until 1980 [1]. This was the first identified antibiotic efflux transporter, a metal-tetracycline/Hþ antiporter [5], which belongs to a major facilitator superfamily (MFS) [6]. Since then, many homologues of tetracycline exporters have been found and classified into class A–H, and K–L [7]. Most are encoded in R-plasmids. Current Opinion in Chemical Biology 2003, 7:570–579
Although some catalyze Naþ and Kþ exchange with Hþ [8], no physiological substrate has been found. The structure and function of tetracycline/Hþ antiporter has been thoroughly studied and thus it has become almost the paradigm for molecular biological studies of drug/Hþ antiport mechanisms. The hydrophobic and metastable nature of membrane transport proteins has long prohibited the crystal structure determination, until a recent breakthrough with ABC transporters [9,10] and a multidrug efflux transporter [11]. Very recently, Kaback et al. and Lemieux et al. solved the crystal structures of MFS-type secondary transporters in Escherichia coli — lac permease and glycerol-3-phosphate transporter, respectively (personal communication). However, the situation is still unaltered in many other transporters, including drug exporters. In such cases, site-directed mutagenesis and chemical modification studies are useful alternatives to investigate the structure and mechanism of membrane transport [12,13–15]. Recent crystallographic analysis has corroborated the structure of AcrB, which was estimated by site-directed chemical modification [11]. As cysteine-scanning mutagenesis is especially useful for structural and functional studies on membrane transporters, many have been studied using this technique, including lac permease [16], P-glycoprotein [17,18], glucose transporter [19,20], glutamate transporter [21,22], prostaglandin transporter [23] and mitochondrial oxoglutarate transporter [24]. With respect to drug exporters, complete cysteine-scanning mutants of 400 amino acid residues of tetracycline/Hþ antiporter TetA(B) were constructed and extensively analyzed [25]. Here we briefly review the structural and functional implications of the studies on the complete cysteine-scanning mutants of TetA(B).
Metal-tetracycline/HR antiport Tetracycline enters cells by simple diffusion through the lipid bilayer region of the plasma membrane as a protonated neutral form (Figure 1) [26]. Then, in the cell interior, it loses a proton and chelates with Mg2þ. The resulting monovalent cation is exported by TetA(B) coupled with proton influx (Figure 1) [5]. Thus TetA(B) functions as a metal–tetracycline/Hþ antiporter.
Membrane topology Although crystal structures of membrane proteins are notoriously elusive [27], the transmembrane structures themselves are rather simple, that is, they mostly comprise www.current-opinion.com
Mechanisms of drug/HR antiport Tamura, Konishi and Yamaguchi 571
Figure 1
Figure 2
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Tetracycline transport cycle in resistant E. coli cells. (a) Chemical structure of tetracycline. W1–4 represent water molecules. (b) Tetracycline influx and TetA(B)-mediated efflux in E. coli. TH2, TH and THMgþ indicate a neutral form, a monoanionic form, and a Mg2þ chelate complex of tetracycline, respectively.
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a-helix bundle [28]. Therefore, if we can determine the numbers and the ranges of the transmembrane segments and their arrangement in the membrane plane, we can roughly estimate the membrane protein structure. The first step is to determine the membrane-spanning topology. The most popular method is by reporter-enzyme fusion [29,30]. However, it is questionable whether the fusion proteins reflect their native structures. We have developed an alternative method based on the site-directed competitive chemical modification of cysteine-introducing mutants into a cysteine-free derivative of a membrane protein [31] (Figure 2a,b). In this method, two kinds of maleimide derivative, membrane-permeable N-ethyl maleimide (NEM) and membrane-impermeable 4-acetamido40 -meleimidylstilbene-2,20 -disulfonic acid (AMS), are used. The inside/outside localization of introduced www.current-opinion.com
Determination of the topology of tetracycline/Hþ antiporter TetA(B) by site-directed competitive chemical modification of Cys mutants. (a) Principle of the determination of the inside/outside/embedded location of cysteine residues. (b) An example of [14 C] NEM binding to Cys mutants introduced to the periplasmic, cytoplasmic and membrane regions and the competitive inhibition with AMS pretreatment. Labeling was performed in intact cells followed by preparation of the membrane. Radioactive bands were detected by autoradiography of the SDS electrophoresis gel. (c) Topology determined by the competitive labeling. Open and closed ovals indicate the Cys-introduced positions of which NEM binding was not affected and prevented by AMS pretreatment, respectively.
cysteine residues is judged from competitive binding of these two compounds in intact E. coli cells expressing Cys mutants. This method is quite reliable as Cys mutants retaining the drug transport activity can be used for the Current Opinion in Chemical Biology 2003, 7:570–579
572 Mechanisms
Figure 3
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experiment. The results are shown in Figure 2c, indicating that TetA(B) has 12 transmembrane segments [31]. The same method has been applied to topology determination of a multidrug transporter AcrB [32] and a macrolidespecific ABC-type exporter MacB [33]. The exactness of the estimation by this method was later supported by X-ray crystal structure determination of AcrB [11].
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Current Opinion in Chemical Biology 2003, 7:570–579
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If such a water-filled channel exists at the center of the TetA(B) molecule, why doesn’t TetA(B) cause uncoupling of the membrane? To answer this question, we investigated the accessibility of membrane-impermeable maleimide AMS to the NEM-reactive Cys mutants in the transmembrane region. For example, in the case of TM5, AMS prevented NEM binding to G150C, G149C, P146C and G145C but does not affect the binding to L142C,
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Therefore, we replaced all 400 amino acid residues of TetA(B), except for the first methionine, with cysteine one by one [25]. It was revealed that TM5, TM8 and TM11 face a water-filled channel similar to TM2. For TM1, TM4, TM7 and TM10, the N-terminal halves are embedded in the membrane and the C-terminal halves face the water channel (Figure 4a). Because oddnumbered helices penetrate the membrane in the N to C direction while even-numbered ones penetrate in the C to N direction TM1 and TM7 face the water channel at their periplasmic halves, and TM4 and TM10 face it at their cytoplasmic halves. Thus, the channel wall is composed of TM1, TM2, TM5, TM7, TM8 and TM11 in the periplasmic half and TM2, TM4, TM5, TM8, TM10 and TM11 in the cytoplasmic half (Figure 4b).
L88C
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However, with TM2, the Cys-scanning mutants did not show the continuous NEM-non-reactive region (Figure 3b). Instead, the transmembrane region showed periodical appearance of NEM-reactive and non-reactive positions. The NEM-reactive positions are located on the same side of the a-helix, indicating that TM2 faces the water-filled channel (Figure 3c). This suggests that we can distinguish the water-channel-facing helices and the membrane-embedded helices.
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Because the reactivity of a maleimide compound with a sulfhydryl group is very low in the hydrophobic environment, it is possible to determine the exact range of a membrane-spanning region using NEM reactivity with Cys mutants. Figure 3a shows the typical [14 C] NEMlabeling results of cysteine-scanning mutants around transmembrane segment 3 (TM3) [34]. A 20 residuelong continuously NEM non-reactive region was observed in TM3, indicating the membrane-embedded transmembrane a-helix. The same continuously NEMnon-reactive regions were also observed in TM6 [35], TM9 [36] and TM12 [25]. A similar observation was also reported for EmrE [37].
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Current Opinion in Chemical Biology
[14 C] NEM labeling of the cysteine-scanning mutants of TetA(B). (a) Labeling around TM3 and its flanking regions. The underline indicates the predicted TM3 region. (b) Labeling around TM2 and its flanking regions. The underline indicates the predicted TM2 region. (c) 3D model of predicted TM2. Closed ovals with outlined letters indicate the NEM reactive positions. White ovals indicate the NEM nonreactive positions. The gray arrow is a predicted water-filled channel.
G139C and F138C in intact cells (Figure 4a), while AMS prevents NEM binding of all these Cys mutants in sonicated membrane fragments [25]. These observations www.current-opinion.com
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Figure 4
Results of competitive labeling of the complete cysteine-scanning mutants of TetA(B) with maleimide compounds. (a) Determined topology of TetA(B). Blue and red letters indicate the NEM-reactive and non-reactive positions, respectively. Pink and sky blue regions of transmembrane regions indicate the predicted membrane-embedded and water-exposed regions, respectively. Yellow and white circles indicate the positions at which NEM binding is prevented and not affected, respectively, by AMS pretreatment in intact cells. Green horizontal lines indicate the predicted permeability barrier. (b) Predicted arrangements of transmembrane a-helix wheels at cytoplasmic and periplasmic sides. In each wheel, amino acid residues only in the cytoplasmic or periplasmic half of the transmembrane helix are depicted. Amino acid residues on purple and white backgrounds indicate the NEM reactive and non-reactive positions, respectively.
indicate that the permeability barrier against AMS molecules exists at the middle of TM5. Because AMS can access all of the ‘hidden Cys residues’ in sonicated membrane fragments, the permeability barrier is very thin with the thickness of about one a-helical turn. www.current-opinion.com
Similar barriers were also observed in all channel-facing helices. In addition, the positions of barriers in all helices were located at almost the same membrane depth except for TM1 (Figure 4a). In TM1, the barrier position is near the periplasmic end, suggesting that the correct TM1 Current Opinion in Chemical Biology 2003, 7:570–579
574 Mechanisms
position might be shifted toward the cytoplasmic direction. The AMS competition experiment clearly indicates the presence of the permeability barrier having one a-helical turn thickness at the middle of the waterfilled channel. This permeability barrier keeps TetA(B) from uncoupling the membrane.
Next, we constructed all combinations of double Cys mutations at the cytoplasmic and periplasmic ends of the transmembrane segments. Figure 5a shows the disulfide linkages on the topology model. Figure 5b,c show the putative helix arrangements at the cytoplasmic and periplasmic surface, respectively, with disulfide linkages. At the cytoplasmic side, TM4 formed disulfide linkages with TM8, TM10 and TM11. TM10 formed linkages with TM4 and TM2. However, at the periplasmic side, TM4 and TM10 did not form disulfide linkages with any other helices. Instead, TM1 and TM7 formed disulfide linkages with each other and with other helices at the periplasmic side. Conversely, TM2, TM8 and TM11 formed disulfide linkages at both sides. These observations indicate that the channel-facing helices are changed between periplamic and cytoplasmic sides. TM4 and 10 face the water channel at the cytoplasmic side, whereas
Helix packing To determine the helix arrangement in the membrane plane, we adopted a method based on the detection of the intramolecular disulfide linkage of double Cys mutants. The formation of a disulfide linkage was detected by the band shift on SDS polyacrylamide gel electrophoresis after oxidation with Cu2þ/o-phenanthroline. It was revealed that the helix bundle of TetA(B) is a circular form, that is, the first and last helices are close to each other [38].
Figure 5
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Disulfide cross-linking results. (a) Disulfide cross-linking is shown on the topology of TetA(B). Cysteine-introduced positions are depicted by bold letters. Disulfide cross linking on the cytoplasmic and periplasmic sides are depicted by red and green lines, respectively. (b) Helix arrangement of TetA(B) at cytoplasmic side based on cross-linking results. (c) Helix arrangement at periplasmic side. (d) Helix arrangement at periplasmic side when tetracycline binds to TetA(B). Red lines indicate the disulfide cross-linking. Red broken lines indicate the disulfide cross-linking disappeared in the tetracycline-binding form. Current Opinion in Chemical Biology 2003, 7:570–579
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TM1 and 7 face at the periplasmic side. These observations coincide with the results of NEM accessibility (Figure 4b). Similar helix arrangement and the exchange of the water-channel-facing transmembrane helices are also observed in the oxalate transporter using electron microscopy analysis of two-dimensional crystals [39].
affect the transport activity of the wild-type TetA(B), the S65C mutant is completely inactivated by NEM [43]. Among 36 cysteine-scanning mutants between K63C and Y98C, there are only two Cys mutants, S65C and L97C, that are inactivated by NEM [44]. Interestingly, these hot spots are symmetrically located across TM3.
Significant alteration in the cross-linking pattern was observed when tetracycline bound to TetA(B). As shown in Figure 5, in the presence of tetracycline, TM1 did not form a cross-link with TM7 or TM11 in the periplasmic side. TM2 also did not form a cross-link with TM7, whereas cross-links with TM8, TM11 and TM12 were unaltered. These observations suggest that substrate binding might cause TM1 to move away from TM7/11, resulting in the enlargement of the periplasmic opening of the channel.
Surprisingly, the only functionally essential residues in this motif are Gly62 and Arg70, in addition to Asp66. The mutants of Arg67, Gly69 and Arg71 retain the transport activity [45]. In addition, triple mutants (K63L/R67L/ R71L) also retain the transport activity, indicating that the polycationic nature of this loop is not necessary for the function. The general notion that conserved residues are functionally important might have to be changed.
Possible gate for substrate entering into the exporter Tetracycline/Hþ antiporters have a conserved sequence motif, 62 GXXXDRXGRR71 , in cytoplasmic loop2–3 between TM2 and TM3 (Figure 6), which is also common to MFS transporters [40]. Although TetA(B) has seven conserved acidic residues in the hydrophilic loop region, Asp66 in this motif is the only essential residue of which mutations to any other amino acid residues result in complete loss of transport function [41]. Ser65 is not conserved and its mutation to cysteine did not affect the function; however, position 65 is a hot spot for sulfhydryl inactivation [42]. Although NEM does not
With respect to Arg70, R70K retains some activity but any other mutants are inactive; however Hg2þ or Co2þ can restore the transport activity of R70C via mercaptide formation [46]. These observations indicate that positive charge at position 70 is essential. This positive charge works via ion pairing with a negative charge of Asp120 located in the next loop between TM4 and TM5 [47]. D120N mutation partially restores the activity of the R70A mutant, while D66A mutation has no such effect [47]. The charge exchange mutant R70D/D120R also shows transport activity, although R70D has no activity. The R70C/D120C double mutant also retained activity slightly lower than that of the D120C single mutant with activity significantly higher than that of the R70C single mutant. However, the R70C/D120C double mutant is
Figure 6
Cytoplasm COOH
TM1
D R X G X D66 R R70 S65
G332S R
G X
X
NH2
D120
G62L TM2
TM4
TM3
TM5
TM6
TM7 A220
D15
TM8
TM9
TM10
TM11
TM12
H257 D285
D84 R101 A354D
L30S L97
Periplasm Current Opinion in Chemical Biology
Functionally essential or important residues described in the text are shown on the TetA(B) topology. Pink and blue ovals indicate acidic and basic residues, respectively. White rectangles indicate hot spots for inactivation of Cys mutants with NEM. Red and black rectangles indicate the first mutation sites resulting in loss of resistance and the second mutation sites that suppress the effect of the first mutation, respectively. The dotted line indicates the ion pair. The MFS motif is shown on loop2–3. www.current-opinion.com
Current Opinion in Chemical Biology 2003, 7:570–579
576 Mechanisms
Figure 7
III
I V VIII
IV II
R70 D120 D66
IX
loop2-3
VII
loop4-5
D15
D8
H257
D285
4
0 22
A
R101
Water-filled channel
H+
Current Opinion in Chemical Biology
Postulated model for tetracycline/Hþ antiport mediated by TetA(B). In this model, TM6, 10, 11 and 12 are removed for easy inspection. Metaltetracycline complex enters into TetA(B) through the gate composed of loop2–3 and loop4–5. Then it is transported through the water-filled channel, probably close to the wall composed of helices 1, 2 and 4. Finally, it is released from TetA(B) near Arg101. Helix 3 is embedded in the membrane, while Asp84 may be exposed by substrate-induced conformational change during transport function. The proton translocation pathway might be separated from the substrate pathway and located in the hydrophobic core composed of helices 7, 8 and 9.
completely inactivated with Hg2þ, probably because the side chains are bridged with Hg2þ, indicating that the dynamic association/dissociation of the Arg70–Asp120 ion pair is required for tetracycline transport function. These observations suggest that loop2–3 may function as a gate for tetracycline entry into TetA(B) (Figure 7).
Functional charged residues in the transmembrane region In the transmembrane region of TetA(B), there are three acidic residues (Asp15, 84 and 285) and two basic residues (Arg101 and His257). These five charged residues are highly conserved in tetracycline exporters [48–50]. Sitedirected mutagenesis studies revealed that all of them are functionally important [48,49,51] (Figure 6). Out of three acidic residues, Asp285 is essential for TetA(B) function. When it is replaced with any other residues, the resulting mutant lost not only tetracycline transport activity but also the ability for tetracycline binding [52]. Asp285 is located at the middle of TM9, a fully membrane-embedded transmembrane a-helix [36]. His257 is located at the middle of TM8 and plays an important role in proton Current Opinion in Chemical Biology 2003, 7:570–579
translocation [50] probably via ion pairing with Asp285. We found a fascinating second-site suppressor mutant from D285N mutant [53]. In that mutant, Ala220 was replaced with Glu. The resulting mutant recovered almost full drug resistance and about half of tetracycline export activity. Ala220 is located in TM7 at almost the same membrane depth as His257 and Asp285. These observations might implicate that the triplet of TM7, TM8 and TM9 forms a proton translocation pathway (Figure 7). Arg101 is another essential charged residue in the transmembrane region and is located near the periplasmic end of TM4 [49]. Considering the symmetry of sulfhydryl hot spots, S65C in loop2–3 and L97C in loop3–4, Arg101 may play a similar role to Arg70 in loop2–3, as an exit gate for substrate translocation (Figure 7).
Second-site suppressor mutations indicate structural and functional coupling between separated residues TetA(B) gave various second-site suppressor mutations other than D285N/A220E, as described above. The www.current-opinion.com
Mechanisms of drug/HR antiport Tamura, Konishi and Yamaguchi 577
typical one is the L30S mutation, which recovers drug resistance from the G62L mutant (Figure 6) [54]. Gly62 is located in cytoplasmic loop2–3 and is the first residue of the MFS conserved motif [40]. Leu30 is located at the opposite side in loop1–2. Interestingly, when NEM accessibility to L29C is used for a measure of the local conformation around loop1–2, position 29 is exposed to [14 C] NEM in the wild-type TetA(B) having only the L29C mutation, which is neutral for the transport function. However, position 29 is hidden in G62L mutant, indicating that the effect of the Gly62Leu mutation in cytoplasmic loop2–3 is transmitted to the periplasmic loop1–2 to hide position 29. However, the second-site Leu30Ser mutation recovers the effect of G62L and exposes position 29 to the medium. Such remote conformational linkage is the cause for recovery in drug resistance. Similar transmembrane conformational linkage is also observed across TM11. That is, the loss of activity with Gly332Ser mutation in loop10–11 is recovered by Ala354Asp mutation in loop11–12 (Figure 6) [55]. This is also due to the occlusion of position 354 with G332S mutation and exposition again with A354D second-site mutation [55]. Interestingly, G332S mutation is also suppressed by L30S mutation and G62L mutation is suppressed by A354D mutation, indicating that TM2 and TM11 are close to each other and conformationally linked [55], which is supported by the helix arrangement estimated by disulfide cross-linking (Figure 5).
results suggest that proton translocation and substrate transport are separated and only conformationally linked. This is consistent with the recent crystallographic study of lac permease, in which the substrate pathway and proton translocation pathway are separated by at least 6 A˚ . RND-type multidrug exporter AcrB is also driven by remote-conformational coupling [11]. Therefore, the remote-conformational coupling between proton translocation and the substrate transport may be a general rule in a secondary transporter.
Conclusion Site-directed mutagenesis and the chemical modification studies on tetracycline/Hþ antiporter TetA(B) as a model for drug/Hþ antiport revealed the presence of a central water-filled channel with a thin permeability barrier at the middle of the channel. As the distances between transmembrane helices across the channel are increased at the periplasmic side when tetracycline binds to TetA(B), the channel acts as at least a part of the tetracycline translocation pathway. However, some residues important or essential for substrate translocation are located in the membrane-embedded face of the helices (Figure 7), suggesting the conformational change and substrate-induced exposure of these residues. Our results indicate that tetracycline enters TetA(B) through a gate between loop2–3 and loop4–5. It is then transported through the water-filled channel, probably close to the wall composed of N-terminal helices such as TM1, TM2 and TM4. Finally it is released from an exit gate around Arg101. TM3 is embedded in the membrane, while Asp84 may be exposed by substrate-induced conformational change during transport function. The residues important for proton translocation are mainly located on the membrane-embedded surface of the C-terminal helices. The proton translocation pathway seems to be separated from the water-filled channel and the substrate translocation pathway (Figure 7). Our www.current-opinion.com
Acknowledgements We thank Tomomi Kimura-Someya, Yuichi Someya, and past and present members of our laboratory.
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