The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism
2000; Springer Nature; Volume: 19; Issue: 11 Linguagem: Inglês
10.1093/emboj/19.11.2503
ISSN1460-2075
Autores Tópico(s)DNA and Nucleic Acid Chemistry
ResumoArticle1 June 2000free access The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism Hendrik W. van Veen Corresponding Author Hendrik W. van Veen Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands Search for more papers by this author Abelardo Margolles Abelardo Margolles Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands Search for more papers by this author Michael Müller Michael Müller Department of Internal Medicine, Division of Gastroenterology and Hepatology, University Hospital Groningen, Hanzeplein 1, NL-9713 EZ Groningen, The Netherlands Search for more papers by this author Christopher F. Higgins Christopher F. Higgins MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN UK Search for more papers by this author Wil N. Konings Wil N. Konings Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands Search for more papers by this author Hendrik W. van Veen Corresponding Author Hendrik W. van Veen Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands Search for more papers by this author Abelardo Margolles Abelardo Margolles Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands Search for more papers by this author Michael Müller Michael Müller Department of Internal Medicine, Division of Gastroenterology and Hepatology, University Hospital Groningen, Hanzeplein 1, NL-9713 EZ Groningen, The Netherlands Search for more papers by this author Christopher F. Higgins Christopher F. Higgins MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN UK Search for more papers by this author Wil N. Konings Wil N. Konings Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands Search for more papers by this author Author Information Hendrik W. van Veen 1, Abelardo Margolles1, Michael Müller2, Christopher F. Higgins3 and Wil N. Konings1 1Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands 2Department of Internal Medicine, Division of Gastroenterology and Hepatology, University Hospital Groningen, Hanzeplein 1, NL-9713 EZ Groningen, The Netherlands 3MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2503-2514https://doi.org/10.1093/emboj/19.11.2503 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters. Introduction Multidrug resistance in pro- and eukaryotic cells is often associated with the enhanced expression of multidrug transport proteins (van Veen and Konings, 1997). A protein in Lactococcus lactis, LmrA, mediates drug and antibiotic resistance by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane (Bolhuis et al., 1996; van Veen et al., 1996). LmrA is a structural and functional homolog of the mammalian multidrug resistance P-glycoprotein, overexpression of which is one of the major causes of resistance of cancers to chemotherapy (Gottesman et al., 1995). Bacterial LmrA can substitute for P-glycoprotein in human lung fibroblast cells, and both proteins have a very similar drug and modulator specificity (van Veen et al., 1998). LmrA and P-glycoprotein are both members of the ATP-binding cassette (ABC) superfamily of proteins (Higgins, 1992). ABC transporters form one of the largest of all protein families with substrate specificities ranging from the uptake of sugars, amino acids and organic ions to the export of large protein toxins. In eukaryotic cells, ABC transporters serve a variety of physiological roles, many of which are associated with disease phenotypes, including the TAP peptide transporter required for Class I antigen presentation, the transporters associated with adrenoleukodystrophy and Zellweger's syndrome, the multidrug resistance-associated (MRP) proteins, sulfonylurea receptors (SUR), the bile salt transporter (BSEP) in hepatocytes, and the ABC1 transporter associated with Tangier disease and familial high-density lipoprotein deficiency (Klein et al., 1999). Most ABC transporters have a common domain organization with four core domains (Higgins, 1992). Two of these domains, the transmembrane domains, are hydrophobic and span the membrane multiple times. There is now compelling evidence that each transmembrane domain of P-glycoprotein consists of six transmembrane segments (putative α-helices), giving a total of 12 transmembrane segments per P-glycoprotein molecule (Kast et al., 1996; Loo and Clarke, 1999a). These domains are thought to form the pathway through which the solute crosses the membrane and confer substrate specificity through one or more substrate-binding sites. The other two domains bind and hydrolyze ATP and couple the energy of ATP hydrolysis to the transport process. These ABC domains, in particular, exhibit significant sequence identity between ABC transporters irrespective of their phylogenetic origin or substrate specificity. P-glycoprotein, like many eukaryotic ABC transporters, has all four domains fused into one single polypeptide, and there is evidence that this monomeric, four-domain protein is the functional unit (Higgins et al., 1997; Rosenberg et al., 1997; Loo and Clarke, 1999a). The protein appears to have arisen by a gene duplication event, fusing two related half-molecules each consisting of one ABC domain and one transmembrane domain. In contrast, LmrA appears to be a half-transporter consisting of an N-terminal transmembrane domain with six membrane-spanning segments, fused to an ABC domain. It has been predicted that LmrA functions as a homodimer to form a full transporter with four core domains (van Veen et al., 1996); we show in this paper that this is indeed the case. P-glycoprotein and LmrA utilize the free energy of ATP to pump drugs out of the cell. The two proteins exhibit a basal rate of ATP hydrolysis that is stimulated 3- to 4-fold in vitro by transport substrates, and have a similar apparent affinity for Mg-ATP (Senior et al., 1995; Martin et al., 1997; van Veen et al., 1998). For P-glycoprotein, both the ABC domains have been shown to bind and hydrolyze ATP (Hrycyna et al., 1998), and several observations provide strong support to an alternating catalytic sites model in which the ABC domains of P-glycoprotein act alternately to hydrolyze ATP: (i) mutations (Urbatsch et al., 1998) or covalent modifications (Senior and Bhagat, 1998; Loo and Clarke, 1999a) that inactivate ATP hydrolysis by one of the ABC domains abolish all drug-stimulated ATPase activity, demonstrating catalytic cooperativity between the two ABC domains of P-glycoprotein; (ii) o-vanadate, a potent inhibitor of P-glycoprotein and LmrA-associated ATPase and drug transport activities, permits only a single turnover of P-glycoprotein by trapping either the first or the second ABC domain in a catalytic transition state conformation, thus preventing the other ABC domain from doing so (Urbatsch et al., 1995); and (iii) photoaffinity labeling experiments with azido-nucleotides suggest that ATP binding to one ABC domain stimulates ATP hydrolysis at the other ABC domain (Ueda et al., 1999), and that 1 mol of Mg-8-azido-ADP is bound per mol of P-glycoprotein after hydrolysis of Mg-8-azido-ATP (Senior et al., 1995). However, the mechanism by which P-glycoprotein and LmrA couple the hydrolysis of ATP to the movement of drugs across the membrane is not known. Indeed, the number and nature of the drug-binding site(s) are also ill-defined. There is strong evidence that the transmembrane domains of P-glycoprotein form the binding site(s) for transported drugs (Ueda et al., 1997; Loo and Clarke, 1999b). Genetic analysis has revealed that replacement of a large number of individual amino acids can affect drug specificity (Gottesman et al., 1995), but since these amino acids are scattered throughout the transmembrane segments and intervening loops, genetic analysis has failed to define a drug-binding pocket. Photoaffinity labeling studies with drug analogs have indicated two regions within the transmembrane domains of P-glycoprotein encompassing transmembrane segments 5 and 6 in the N-terminal half, and transmembrane segments 11 and 12 in the C-terminal half (Greenberger, 1993; Morris et al., 1994), which may form part of one large drug interaction domain, or which may represent two or more distinct drug-binding sites (Dey et al., 1997). Owing to the lack of specificity of the cross-linking reaction, photoaffinity labeling has been unable to identify the amino acid residues involved in drug binding. The notion that P-glycoprotein contains at least two drug-binding sites is supported by studies employing continuous fluorescence measurements of P-glycoprotein-mediated drug transport in plasma membrane vesicles (Shapiro and Ling, 1997). In addition to binding sites for transported drugs, detailed pharmacological analysis shows at least one binding site in P-glycoprotein and LmrA for allosteric modulators such as the indolizin sulfone SR33557 (Martin et al., 1997) and the 1,4-dihydropyridine nicardipine (Ferry et al., 1992; van Veen et al., 1998) that are bound to, but not transported by the protein. In this study we have carried out a detailed analysis of the drug-binding sites of LmrA by transport measurements, vinblastine equilibrium binding determinations, and photoaffinity labeling using N-(4′,4′-azo-n-pentyl)-21-deoxy-[3H]ajmalinium (APDA) (Müller et al., 1994; van Veen et al., 1996). The results demonstrate the presence of two vinblastine-binding sites: a low-affinity binding site exposed at the outside (extracellular) surface of the membrane, which is allosterically coupled to a high-affinity binding site exposed at the inside (intracellular) surface of the membrane. Only the low-affinity, outside-facing vinblastine-binding site is accessible in the vanadate-trapped transporter. The data suggest an alternating two-site mechanism in which the transport protein oscillates between two configurations, each containing a high-affinity, inside-facing, transport-competent drug-binding site, and a low-affinity, outside-facing drug-release site. The ATP-dependent interconversion of one configuration into the other proceeds via a catalytic transition state conformation in which the transport-competent drug-binding site is occluded. This model is analogous to a two-cylinder engine, and has implications for the mechanism of action of P-glycoprotein and other ABC transporters. Results LmrA and P-glycoprotein have an identical domain organization From its primary amino acid sequence, LmrA appears to be a 'half' ABC transporter consisting of one transmembrane domain and one ATP-binding domain. To assess whether two LmrA molecules cooperate to form a single transporter, the functional properties of defined covalently linked dimers of LmrA were studied. Two copies of the lmrA coding sequence were fused, in frame, connected by a linking sequence identical to the 'linker' region that connects the two halves of P-glycoprotein. The coding sequence for this LmrA dimer was cloned into the lactococcal expression vector pNZ8048 under the control of the nisin-inducible nisA promoter, which has previously been used for the expression of LmrA (Margolles et al., 1999). In addition to the wild-type dimeric LmrA protein (designated KK), single mutant dimeric LmrA proteins were generated in the same vector by introducing lysine to methionine substitutions (van Veen et al., 1998) in the Walker A region of either the N- or the C-terminal ABC domain of the dimer (designated MK and KM, respectively). A double mutant was also generated with a lysine to methionine substitution in both ABC domains (designated MM). His6 tags were placed at the N-termini of each of the proteins. Upon the addition of 10 ng/ml nisin to L.lactis cells containing plasmids encoding the 'dimeric' lmrA genes, equivalent amounts of a 132 kDa polypeptide could be detected using an anti-His6 tag monoclonal antibody (Figure 1A). Wild-type LmrA is able to reduce the accumulation of ethidium bromide in the cell by mediating its efflux (van Veen et al., 1996). Measurements of ethidium bromide accumulation in non-expressing control cells, cells expressing wild-type LmrA and cells expressing the KK form of dimeric LmrA demonstrated that the KK form (having wild-type nucleotide-binding domains) was transport active (Figure 1B). The difference in ethidium accumulation between cells containing monomeric LmrA or dimeric (KK) LmrA was consistent with a difference in the level of expression of the two proteins [∼10% of total membrane protein for LmrA compared with 2% of total membrane protein for dimeric (KK) LmrA; see the later section 'Direct determination of LmrA:vinblastine binding stoichiometry' for experimental details]. In contrast, the MK, KM and MM mutant forms of dimeric LmrA had all lost the ability to transport ethidium from the cell. Cells expressing these mutant forms displayed a similar ethidium accumulation level to that observed in non-expressing control cells, which lack significant LmrA activity. Figure 1.Expression and activity of covalently linked dimers of LmrA. (A) Western blot of total membrane protein (20 μg) from inside-out membrane vesicles lacking LmrA (lane 1), or containing LmrA (lane 2), the wild-type (KK) form of dimeric LmrA (lane 3), or the dimeric form of LmrA with a K to M substitution in the Walker A region of the first ABC domain (MK, lane 4), the second ABC domain (KM, lane 5), or both ABC domains (MM, lane 6). The western blot was probed with an anti-His6 tag antibody. The migration of molecular mass markers is indicated. An arrowhead indicates the position of monomeric LmrA (66 kDa) and dimeric LmrA (132 kDa). (B) Accumulation of ethidium bromide in cells expressing LmrA, or the KK, MK, KM or MM forms of dimeric LmrA. Cells were pre-energized for 3.5 min in the presence of 20 mM glucose, after which 2 μM ethidium bromide was added. Ethidium accumulation in the cells was measured by fluorimetry. The uptake of ethidium in control cells not expressing LmrA was similar to that in cells expressing the KM, MK or MM form of dimeric LmrA (not shown). (C) Vanadate-sensitive ATPase activity in inside-out membrane vesicles lacking LmrA (control), or containing LmrA, or the KK, MK, KM or MM form of dimeric LmrA. The ATPase activity was measured at a Mg-ATP concentration of 1 mM in the presence of 20 μM verapamil. Download figure Download PowerPoint LmrA displays a basal ATPase activity, which is stimulated by verapamil and inhibited by o-vanadate (van Veen et al., 1998). Analysis of the verapamil-stimulated, LmrA-associated ATPase activity in inside-out membrane vesicles of L.lactis demonstrated that the KK form of dimeric LmrA had essentially wild-type ATPase activity, whereas the KM, MK and MM mutant forms had lost all LmrA-associated ATPase activity (Figure 1C). Thus, KM, MK and MM mutants of dimeric LmrA have similar properties to the KM, MK and MM mutants of P-glycoprotein, which lack all ATPase and transport activity (Müller et al., 1996; Loo and Clarke, 1999a). These data are consistent with the notion that LmrA functions as a homodimer, analogous to monomeric P-glycoprotein, with both halves of the dimer required for transport. The dimeric nature of the LmrA transporter was also studied by the co-reconstitution into proteoliposomes of two different LmrA proteins: the cysteine-less wild-type LmrA and a mutant form of LmrA in which the N-ethylmaleimide (NEM)-reactive glycine to cysteine (G386C) substitution in the Walker A region (Loo and Clarke, 1999a) was introduced by site-directed mutagenesis. The wild-type and mutant forms were purified by Ni2+-nitrilotriacetic acid (NTA) affinity chromatography, mixed at molar ratios of 6:0, 3:3, 2:4 and 0:6, respectively, and reconstituted into n-dodecyl-β-D-maltoside-destabilized liposomes. After reconstitution, the LmrA-associated transport activity in proteoliposomes was measured using fluorescent 1-myristoyl-2-[6-[(7-nitro-2,1,3-benz-oxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoeth-amine (C6-NBD-PE) (Margolles et al., 1999). In the absence of NEM treatment after the reconstitution procedure, equal rates were observed for the LmrA-mediated transbilayer movement of NBD-PE in proteoliposomes containing the wild-type and mutant proteins at the various ratios (Figure 2). Hence, the single-cysteine mutant form of LmrA had retained its transport activity compared with that of wild-type LmrA. When the proteoliposomes were incubated for 30 min in the presence of 1 μM NEM, allowing NEM to react with the cysteine residue in the G386C mutant form of LmrA, the LmrA-mediated transport of C6-NBD-PE in proteoliposomes containing wild-type LmrA only (wild-type:mutant ratio of 6:0) was not affected; the transport rate after NEM treatment was 98 ± 5% (n = 3) of the transport rate before NEM treatment. In contrast, the transport rate in NEM-treated proteoliposomes containing wild-type and mutant proteins at a ratio of 3:3, 2:4 and 0:6 was reduced to 22 ± 5, 13 ± 4 and 0 ± 4% (n = 3) of the transport rate in proteoliposomes without NEM treatment, respectively. This inhibition pattern suggested that two wild-type LmrA monomers cooperate to form a functional transporter, as 100, 25, 10 and 0% of the dimers would contain two wild-type monomers at a wild-type:mutant ratio of 6:0, 3:3, 2:4 and 0:6, respectively. On the other hand, the observed inhibition pattern is not consistent with the presence of three or four (or more) wild-type LmrA monomers in the minimal functional unit, as 100, 13, 4 and 0% of the trimers would contain three wild-type monomers, and 100, 6, 1 and 0% of the tetramers would contain four wild-type monomers at a wild-type:mutant ratio of 6:0, 3:3, 2:4 and 0:6, respectively. These data are in agreement with the electron microscopy analysis of purified and functionally reconstituted LmrA, which revealed small, uniform particles with a diameter of 8.5 nm by 5 nm (S.Scheuring, A.Margolles, H.W.van Veen, W.N.Konings and A.Engel, unpublished data), similar to that previously observed for the monomeric P-glycoprotein (Rosenberg et al., 1997). Taken together, these data demonstrate that the dimeric state of LmrA is a prerequisite for function, analogous to the monomeric P-glycoprotein, and that functional cross-talk between the two LmrA monomers is essential for transport. Figure 2.Negative dominance of an NEM-inactivated single-cysteine LmrA mutant in proteoliposomes containing co-reconstituted wild-type and mutant LmrA proteins. The transport activity in proteoliposomes was measured using fluorescent C6-NBD-PE as a substrate. Inset: typical NBD fluorescence traces obtained for proteoliposomes containing wild-type LmrA in assay mixtures supplemented with ATP or non-hydrolyzable AMP-PNP demonstrate the experimental and analytical methods used. At the onset of transport measurement, C6-NBD-PE was present in donor liposomes in which the NBD fluorescence was low due to the presence of N-Rh-PE quencher. Addition of proteoliposomes at the arrow resulted in the movement of C6-NBD-PE, but not of the N-Rh-PE quencher, from donor liposomes to proteoliposomes with a concomitant increase in NBD fluorescence. The increase in NBD fluorescence observed was deconvoluted by a double exponential fit of the data (see Materials and methods), which is shown displaced from the fluorescence traces for clarity, yielding a rate constant k1 for the interbilayer movement of C6-NBD-PE between donor liposomes and proteoliposomes, and a rate constant k2 for the transbilayer movement of the probe in the proteoliposomes. Main figure: NBD-PE transport was measured in the presence of ATP or AMP-PNP, using proteoliposomes containing the cysteine-less wild-type LmrA and the G386C mutant form of LmrA at the molar ratios indicated, before (filled circles) and after (open circles) NEM treatment of the proteoliposomes. In all experiments k1 values were of the order of 0.038–0.041 s−1, and were at least 10 and 70 times the k2 values obtained for incubations containing ATP or AMP-PNP, respectively. Hence, the transbilayer movement of C6-NBD-PE could be explicitly characterized, separate from the interbilayer movement. The relative transbilayer transport rate, as depicted in the graph, was calculated from the estimated k2 values using the equation: vrel = vATP/vAMP-PNP = k2ATP/k2AMP-PNP. The k2 values obtained for proteoliposomes in incubations supplemented with AMP-PNP were relatively constant, and of the order of 0.00052–0.00058 s−1. Download figure Download PowerPoint Vinblastine and Hoechst 33342 affect the transport of each other via LmrA LmrA and P-glycoprotein are functionally interchangeable proteins with very similar drug and modulator specificities (van Veen et al., 1998). Previously, we have used 2-[2-(4-ethoxyphenyl)-6-benzimidazolyl]-6-(1-methyl)-(4-piperazil)-benzimidazole (Hoechst 33342) and vinblastine to monitor drug transport by LmrA expressed in human lung fibroblast cells (van Veen et al., 1998). Hoechst 33342 is fluorescent when it resides in the lipid environment of the membrane, and is essentially non-fluorescent in the aqueous phase. Transport of Hoechst 33342 from inside-out membrane vesicles of L.lactis by LmrA resulted in a decrease in Hoechst 33342 fluorescence (Figure 3A). Interestingly, the rate of LmrA-mediated Hoechst 33342 transport in inside-out membrane vesicles was enhanced in the presence of low concentrations of vinblastine. In a similar fashion, Hoechst 33342 at low concentrations also stimulated the ATP-dependent accumulation of vinblastine in inside-out membrane vesicles of LmrA-overexpressing L.lactis (Figure 3B). Both drugs inhibited the transport of each other when applied at 10-fold higher concentrations (Figure 3). Under the experimental conditions, vinblastine did not affect the intrinsic fluorescence of Hoechst 33342, and vinblastine and Hoechst 33342 did not affect the partitioning of each other in the phospholipid bilayer (data not shown; Putman et al., 1999). The observation that vinblastine and Hoechst 33342 are able to stimulate the transport of each other by LmrA at low drug concentrations is not readily explained if the LmrA transporter contains only a single drug-binding site. Instead, these data and the reciprocal inhibition of transport at high drug concentrations suggest at least two binding sites with overlapping drug specificities, which are functionally coupled. Figure 3.LmrA-mediated transport of Hoechst 33342 and vinblastine. (A) Effect of vinblastine on the transport of Hoechst 33342 was measured by fluorimetry. After 30 s of pre-incubation of inside-out membrane vesicles containing LmrA in transport buffer, 2 μM Hoechst 33342 was added and its binding to the membrane vesicles followed in time until a steady state was reached. Hoechst 33342 transport was initiated by the addition of 2 mM Mg-ATP. Incubations with Mg-ATPγS served as a control. Vinblastine (2 μM; VBL), added to the membrane vesicles prior to the addition of Hoechst 33342, did not affect Hoechst 33342 fluorescence in experiments with ATPγS (not shown), but significantly stimulated the ATP-dependent transport of Hoechst 33342. In the presence of 20 μM vinblastine, ATP-dependent Hoechst 33342 transport was completely inhibited. The fluorescence trace obtained under these conditions was indistinguishable from that of the depicted control experiment and is therefore not shown. (B) Effect of Hoechst 33342 on the LmrA-mediated transport of [3H]vinblastine. The uptake of vinblastine in inside-out membrane vesicles in buffer supplemented with 40 nM [3H]vinblastine and 2 mM Mg-ATPγS (open squares, open circles, open triangles) or 2 mM Mg-ATP (filled squares, filled circles, filled triangles) was measured in the absence (open and filled circles) or presence of 2 μM Hoechst 33342 (open and filled squares) or 20 μM Hoechst 33342 (open and filled triangles). Download figure Download PowerPoint LmrA contains two non-identical, allosterically linked vinblastine-binding sites The drug-binding sites involved in LmrA-mediated vinblastine transport were studied in more detail by equilibrium binding using [3H]vinblastine. The specific binding of vinblastine to LmrA in inside-out membrane vesicles was plotted as a function of the free vinblastine concentration in the water phase, which could be determined more accurately than the free vinblastine concentration in the phospholipid bilayer, and which increased linearly with the amount of vinblastine associated with the membrane at total vinblastine concentrations up to 1 μM in accordance with a membrane/water partition coefficient of 278 (data not shown). Figure 4A shows that the specific binding of vinblastine to LmrA increased as a function of the free vinblastine concentration in a saturable fashion. No specific binding of vinblastine was observed in inside-out membrane vesicles in the absence of LmrA. The binding data could not readily be fitted to a hyperbolic curve, as shown by the residuals between the experimental data and the best-fit hyperbola as a function of the free vinblastine concentration in the assays (Figure 4B). The hyperbola overestimates the amount of drug binding at vinblastine concentrations below 50 nM, and underestimates binding at vinblastine concentrations between 50 and 160 nM. Similar results were obtained for the KK, KM, MK and MM forms of dimeric LmrA expressed in inside-out membrane vesicles of L.lactis (data not shown). Thus, experimental measurements of vinblastine binding to LmrA are not consistent with a single drug-binding site. Instead, the experimental vinblastine binding data fit a cooperative, two-site drug-binding model: Figure 4.Vinblastine equilibrium binding to LmrA. (A) Specific binding of [3H]vinblastine to inside-out membrane vesicles without LmrA (control) or with LmrA, as a function of the free vinblastine concentration. Superimposed on the data are the best fit curves obtained for a single-site binding model (hyperbolic, dotted curve), the cooperative two-site binding model (sigmoidal, solid curve) and the Hill equation (indistinguishable from the sigmoidal, solid curve). (B) Residual variance between the data and the best fitting hyperbola and sigmoidal curves from (A), plotted as a function of the free vinblastine concentration. Note the systematic deviation of the residual variance of the data for the hyperbola, indicating that the single-site model provides an inadequate fit to the data. The residual variance between the data and the sigmoidal curve shows only random scatter, suggesting that the data are adequately fitted by the cooperative two-site binding model. (C) Heterologous displacement of vinblastine from LmrA by CP100-356. LmrA was saturated with [3H]vinblastine at a concentration of 192 nM, and vinblastine displacement from LmrA by CP100-356 measured at increasing concentrations of CP100-356. The data were fitted by the cooperative two-site drug-binding model in which direct competition was introduced between vinblastine and CP100-356 for binding to the two drug-binding sites in the LmrA transporter. A relative binding value of 1 represents 1.42 nmol vinblastine/mg of membrane protein. (D) Residual variance between the experimental data and the best fitting curve from (C), showing random scatter of the data around the predicted curve. Download figure Download PowerPoint where T is the transport protein and S is a drug molecule. In this model, an initial vinblastine-binding event with low affinity (Kd1) initiates a second vinblastine-binding event with high affinity (Kd2). Figure 4B shows that, for such a cooperative two-site drug-binding model, the residual varianc
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