Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.     

Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.     

Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil

Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a K(m) value (25 microM) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil.     

Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 and 7-10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo, T. W. and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948; Loo, T. W. and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12.     

Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds.     

Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane

Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.     

Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane

The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.     

Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites

Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.     

Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.     

Identification of Residues in the Drug Translocation Pathway of the Human Multidrug Resistance P-glycoprotein by Arginine Mutagenesis

P-glycoprotein (P-gp, ATP-binding cassette B1) is a drug pump that extracts toxic drug substrates from the plasma membrane and catalyzes their ATP-dependent efflux. To map the residues in the drug translocation pathway, we performed arginine-scanning mutagenesis on all transmembrane (TM) segments (total = 237 residues) of a P-gp processing mutant (G251V) defective in folding (15% maturation efficiency) (glycosylation state used to monitor folding). The rationale was that arginines introduced into the drug-binding sites would mimic drug rescue and enhance maturation of wild-type or processing mutants of P-gp. It was found that 38 of the 89 mutants that matured had enhanced maturation. Enhancer mutations were found in 11 of the 12 TM segments with the largest number found in TMs 6 and 12 (seven in each), TMs that are critical for P-gp-drug substrate interactions. Modeling of the TM segments showed that the enhancer arginines were found on the hydrophilic face, whereas inhibitory arginines were located on a hydrophobic face that may be in contact with the lipid bilayer. It was found that many of the enhancer arginines caused large alterations in P-gp-drug interactions in ATPase assays. For example, mutants A302R (TM5), L339R (TM6), G872R (TM10), F942R (TM11), Q946R (TM11), V982R (TM12), and S993R (TM12) reduced the apparent affinity for verapamil by ∼10-fold, whereas the F336R (TM6) and M986R (TM12) mutations caused at least a 10-fold increase in apparent affinity for rhodamine B. The results suggest that P-gp contains a large aqueous-filled drug translocation pathway with multiple drug-binding sites that can accommodate the bulky arginine side chains to promote folding of the protein.The human multidrug resistance P-glycoprotein (P-gp, ATP-binding cassette B1)² is an ATP-dependent drug pump that mediates efflux of a broad range of hydrophobic compounds out of the cell (1). It is expressed in the epithelium of liver, kidney, and gastrointestinal tract and at the blood-brain or blood-testes barrier where it functions to protect us from cytotoxic compounds. It is clinically important because it contributes to multidrug resistance in diseases such as cancer and AIDS (1).P-gp is an ATP-binding cassette transporter of 1280 amino acids that consists of two homologous halves (2). Each half begins with a transmembrane domain (TMD) containing six TM segments followed by a nucleotide-binding domain (NBD).A key goal to understanding the mechanism of P-gp drug transport is to identify the amino acids that line the drug translocation pathway. Because P-gp extracts drug substrates from the lipid bilayer, the drug-binding pocket/drug translocation pathway are predicted to reside in the transmembrane (TM) segments. We previously showed that the TMDs alone were sufficient for drug binding (3). Expression of the TMDs as separate polypeptides showed that both TMD1 and TMD2 were required for binding drug substrate (4). The results of studies utilizing cysteine-scanning mutagenesis and labeling with thiol-reactive drug substrates suggested that all of the TM segments contribute to the drug-binding pocket/drug translocation pathway (reviewed in Ref. 5). The next step is to identify the specific amino acids that line the drug translocation pathway. It is important to identify amino acids that line the drug translocation pathway and to compare whether the biochemical evidence supports a model of P-gp structure in the closed conformation (6) (NBDs close together that was based on the bacterial Sav1866 crystal structure (7)) or the recent crystal structure of mouse P-gp in the open conformation (NBDs far apart) (8). There have been concerns that the mouse P-gp structure may be a non-native structure or in a conformation that exists very transiently (9).Our approach to map the drug translocation pathway has been to use arginine-scanning mutagenesis of the TM segments of a P-gp processing mutant (G251V) that shows partial maturation (∼15% maturation efficiency) (10). Maturation efficiency can be used to detect folding of P-gp in whole cells by monitoring the conversion of P-gp from a core-glycosylated (150 kDa) protein to a mature protein (170 kDa) that contains complex carbohydrate. Because mutant G251V shows partial maturation, we can detect whether an introduced arginine promotes, inhibits, or has a neutral effect on folding. The rationale for using arginine-scanning mutagenesis was that arginine has a large free energy barrier (17 kcal/mol) for insertion into the lipid bilayer because it is highly charged (11). Therefore, introduction of an arginine into a lipid face of the G251V mutant would likely inhibit maturation, whereas an arginine introduced into the aqueous face of the drug translocation pathway would not inhibit maturation of the mutant P-gp.In an initial study on TM1, we demonstrated the feasibility of the approach (10). All arginines introduced into the predicted lipid-facing positions inhibited maturation, whereas those introduced into positions predicted to face the drug translocation pathway did not. A particularly intriguing observation was that some arginines promoted maturation. The residues at these positions were coincidentally at positions identical to those that reacted with thiol-reactive drug substrates in cysteine-scanning mutagenesis studies and were found to be within the drug-binding pocket (10, 12). This suggested that arginine-scanning mutagenesis could be a useful approach for identifying residues in the drug translocation pathway and for determining the orientation of the TM segments in the membrane.Arginines that promote maturation appear to identify positions that are important for P-gp-drug interactions because they appear to mimic drug rescue of P-gp. It was also found that the ability of arginines (such as I306R in TM5) to promote maturation involved global enhancement of P-gp folding rather than simply compensating for a localized mutation (such as G251V) because other processing mutants could also be rescued (12). Because these arginine mutations enhance folding of P-gp in general, they will be described as enhancer rather than suppressor arginines. In this study we performed arginine-scanning mutagenesis on TMs 2–12 of P-gp processing mutant G251V to determine their orientations in the membrane and to identify residues that line the drug translocation pathway.     

Cross-linking of human multidrug resistance P-glycoprotein by the substrate, tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis. Evidence for rotation of a transmembrane helix.

We identified a thiol-reactive substrate, Tris-(2-maleimidoethyl)amine (TMEA), to explore the contribution of the TM segments 6 and 12 of the human multidrug resistance P-glycoprotein (P-gp) during transport. TMEA is a trifunctional maleimide and stimulated the ATPase activity of Cys-less P-gp about 7-fold. Cysteine-scanning mutagenesis of TM12 showed that the activity of mutant V982C was inhibited by TMEA. P-gp mutants containing V982C (TM12) and another cysteine in TM6 were constructed and tested for cross-linking with TMEA. A cross-linked product was observed in SDS-polyacrylamide gel electrophoresis for mutant L339C(TM6)/V982C(TM12). Cross-linking by TMEA also inhibited the ATPase activity of the mutant protein. Substrates such as cyclosporin A, vinblastine, colchicine, or verapamil inhibited cross-linking by TMEA. In the presence of ATP at 37 degrees C, cross-linking of mutant L339C/V982C was decreased. In contrast, there was enhanced cross-linking of mutant F343C(TM6)/V982C(TM12) in the presence of ATP. These results show that cross-linking must be within the drug-binding domain, that residues L339C(TM6)/V982C(TM12) must be at least 10 A apart, and that ATP hydrolysis promotes rotation of one or both TM helices.     

The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment

The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.     

ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein

P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.     

The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium

P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates. Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the drug-binding pocket is accessible to water.     

The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis

Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments.     

Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket

P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.     

Human P-glycoprotein is active when the two halves are clamped together in the closed conformation

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 Å and 20 Å apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 Å) at 0 °C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.     

Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments

P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: characterization of TMs IV-VII and IX-XII and their accessibility to the aqueous translocation pathway

We have determined the accessibility of the Rickettsia prowazekii ATP/ADP translocase transmembrane domains (TMs) IV-VII and IX-XII to the putative, water-filled ATP translocation pathway. A library of 177 independent mutants, each with a single cysteine substitution, was expressed in Escherichia coli, and those with substantial ATP transport activity were assayed for inhibition by thiol-reactive, methanethiosulfonate (MTS) reagents. The MTS reagents used were MTSES (negatively charged), MTSET (positively charged), and MTSEA (amphipathic). Inhibition of ATP transport by a charged MTS reagent indicates the exposure of a TM to the water-filled ATP translocation pathway. The eight TMs characterized in this study had 32 mutants with no assayable transport activity, indicating that cysteine substitution at these positions is not tolerated. ATP transport proficient mutants in TMs IV, V, VII, X, and XI were inhibited by charged MTS reagents, indicating that these TMs are exposed to the aqueous ATP translocation pathway, which is a pattern similar to those of TMs I, II (Alexeyev, M. F. (2004) Biochemistry 43, 6995-7002), and VIII (Winkler, H. H. (2003) Biochemistry 42, 12562-12569). Conversely, ATP-transport-proficient mutants in TMs VI, IX, and XII were not inhibited by charged MTS reagents, indicating that these TMs are sequestered from the aqueous environment, which is a pattern similar to that of TM III (Alexeyev, M. F. (2004) Biochemistry 43, 6995-7002). Preexposure of several MTS-sensitive mutants in TMs V, VII, X, and XI to ATP concentrations 10 times the K(m) resulted in protection from MTS-mediated inhibition; thus, confirming exposure of these TMs to the aqueous ATP translocation pathway, a pattern of protection similar to that observed for TMs I, II, and VIII.     

Do drug substrates enter the common drug-binding pocket of P-glycoprotein through "gates"?

Overexpression of P-glycoprotein (P-gp; ABCB1) can cause multidrug resistance during cancer and AIDS chemotherapy because of its ability to transport a broad range of structurally unrelated compounds from the cell. P-gp is a member of the ABC family of proteins. It is a single polypeptide containing four domains—two transmembrane (TM) domains each of which contains six TM segments and two nucleotide-binding domains. Chemical modification and cross-linking studies of cysteine mutants of P-gp indicate that the common drug-binding pocket is at the interface between the TM domains. It has been postulated that drug substrates enter the lipid bilayer, are extracted by P-gp and transported to the extracellular medium. It is not clear how drug substrates enter the drug-binding pocket. Here, we propose that drug-substrates diffuse from the lipid bilayer into the drug-binding pocket through “gates” formed by TM segments at either end of the drug-binding pocket.