P-glycoprotein misfolds in Escherichia coli : evidence against alternating-topology models of the transport cycle

P-glycoprotein (P-gp) is a drug transporter which pumps toxic hydrophobic compounds out of cells, conferring mutidrug resistance. P-gp is predicted to consist of 12 transmembrane &#102 - helices and there is a strong body of experimental support for this model. However, a number of studies, including those on Pgp expressed in E. coli, have reported topologies with fewer than 12 transmembrane &#102 -helices, leading to the hypothesis that the transmembrane topology of the protein changes during function. It is well established that P-gp undergoes conformational changes during its transport cycle and it has been recently shown that these changes are large in magnitude and could, potentially, reflect a changing transmembrane topology. One therefore, reassessed the transmembrane topology of P-gp expressed in E. coli and compared it directly with the topology ofthe protein express ed in mammalian cells. It was clear that the transmembrane topology of the protein was different in the different cell types and that the misfolding of P-gp in E. coli was due to the misrecognition of multiple P-gp sequences as topogenic signals. Thus, the alternative transmembrane topologies reported for P-gp in E. coli are artefacts of the heterologous expression system used, and models based on such data in which the transmembrane topology changes during drug transport are unlikely to be correct. Instead, the large conformational changes observed during the transportcycle are more likely due to changes in &#102 -helix packing.     

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.     

The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates

Multiple topologies have been detected for the COOH-terminal half of the human multidrug resistance P-glycoprotein (P-gp). In one topology, the predicted third cytoplasmic loop (CL3) is on the cytoplasmic side (P-gp-CL3-cyt) of the membrane. In an alternate topology, CL3 is on the extracellular side of the membrane (P-gp-CL3-ext). It is not known if both forms of P-gp are active because it is difficult to distinguish either topology in the full-length molecule. When the halves of P-gp are expressed as separate polypeptides, the two topologies of the C-Half are readily distinguished on SDS-PAGE, because only the C-Half (CL3-ext) is glycosylated. To test whether both topologies can fold into an active enzyme, we assayed for interaction between the N- and C-Halves of P-gp since functional P-gp requires interaction between both halves. In a mutant P-gp (E875C) that gave about equal amounts of both topologies, only the C-Half (CL3-cyt) could be recovered by nickel chromatography after coexpression with the histidine-tagged N-Half P-gp. The isolated N-Half and E875C C-Half (CL3-cyt) polypeptides, when expressed together, exhibited verapamil- and vinblastine-stimulated ATPase activities that were similar to the wild-type enzyme. We also found that biosynthesis of mutant E875C C-Half in the presence of the N-Half P-gp resulted in enhanced expression of C-Half (CL3-cyt). By contrast, interaction of C-Half (CL3-ext) with N-Half P-gp was not detected. These results show that the topology of the C-Half portion of P-gp greatly influences its interactions with the amino-terminal half of the molecule.     

Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition

Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.     

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.     

Conformational analysis of human ATP-binding cassette transporter ABCB1 in lipid nanodiscs and inhibition by the antibodies MRK16 and UIC2

The human ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp; ABCB1), mediates the ATP-dependent efflux of a variety of drugs. As a result, P-gp plays a critical role in tumor cell drug resistance and the pharmacokinetic properties of most drugs. P-gp exhibits extraordinary substrate and inhibitor promiscuity, resulting in a wide range of possible drug-drug interactions. Inhibitory antibodies have long been considered as a possible strategy to modulate P-gp-dependent cancer cell drug resistance, and it is widely suggested that the antibodies MRK16 and UIC2 inhibit P-gp by capturing a single isoform and preventing flux through the catalytic cycle. Although the crystal structures of many bacterial whole transporters, as well as isolated nucleotide-binding domains, have been solved, high resolution structural data for mammalian ABC transporters are currently lacking. It has been extremely difficult to determine the detailed mechanism of transport of P-gp, in part because it is difficult to obtain purified protein in well defined lipid systems. Here we exploit surface plasmon resonance (SPR) to probe conformational changes associated with these intermediate states for P-gp in lipid bilayer nanodiscs. The results indicate that P-gp in nanodiscs undergoes functionally relevant ligand-dependent conformational changes and that previously described inhibitory antibodies bind to multiple nucleotide-bound states but not the ADP-VO(4)-trapped state, which mimics the post-hydrolysis state. The results also suggest that the substrate drug vinblastine is released at stages that precede or follow the post-hydrolysis ADP-PO(4)·P-gp complex.     

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.     

Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses

The 9 A resolution cryo-electron microscopy map of Sindbis virus presented here provides structural information on the polypeptide topology of the E2 protein, on the interactions between the E1 and E2 glycoproteins in the formation of a heterodimer, on the difference in conformation of the two types of trimeric spikes, on the interaction between the transmembrane helices of the E1 and E2 proteins, and on the conformational changes that occur when fusing with a host cell. The positions of various markers on the E2 protein established the approximate topology of the E2 structure. The largest conformational differences between the icosahedral surface spikes at icosahedral 3-fold and quasi-3-fold positions are associated with the monomers closest to the 5-fold axes. The long E2 monomers, containing the cell receptor recognition motif at their extremities, are shown to rotate by about 180 degrees and to move away from the center of the spikes during fusion.     

Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid

It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E. coli is useful for screening P-gp substrates and inhibitors. In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E. coli UT5600. These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs. Mouse mdr1 was functionally expressed in the most permeable E. coli mutant (UTP17). Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin. To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid. We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance. We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid. This study provide a heterologous system for mdr1 expression in E. coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.     

Translocation mechanism of P-glycoprotein and conformational changes occurring at drug-binding site: Insights from multi-targeted molecular dynamics

P-glycoprotein (P-gp) is well known for multidrug resistance in drug therapy. Its over-expression results into the increased efflux of therapeutic agents rendering them inefficacious. A clear understanding of P-gp efflux mechanism and substrate/inhibitor interactions during the course of efflux cycle will be crucial for designing effective P-gp inhibitors, and therapeutic agents that are non-substrate to P-gp. In the present work, we have modeled P-gp in three different catalytic states. These models were utilized for elucidation of P-gp translocation mechanism using multi-targeted molecular dynamics (MTMD). The gradual changes occurring in P-gp structure from inward open to outward open conformation were sampled out. A detailed investigation of conformational changes occurring in trans-membrane domains (TMDs) during the course of catalytic cycle was carried out. Movements of each TM helices in response to pronounced twisting and translatory motion of NBDs were measured quantitatively. The role of intracellular coupling helices (ICHs) during the structural transition of P-gp was studied, and observed as vital links for structural transition. A close observation of displacements and conformational changes in the residues lining drug-binding pocket was also carried out. Further, we have analyzed the molecular interactions of P-gp substrates/inhibitors during the P-gp translocation to find out how stable binding interactions of a compound at drug-binding site(s) in open conformation, becomes highly destabilized in closed conformation. The study revealed striking differences between the molecular interactions of substrate and inhibitor; inhibitors showed a tendency to maintain stable binding interactions during the catalytic transition cycle.     

Small multidrug resistance proteins: a multidrug transporter family that continues to grow

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane alpha-helices of approximately 100-140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.     

Proline 21, a residue within the α-helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

ΦX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded α-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli . Oligomerization of protein P21G-StrpA was not disturbed.     

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.     

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na(+), Li(+), and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.     

Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans.

The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.     

Molecular genetic and biochemical approaches for defining lipid-dependent membrane protein folding

We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long-range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually dependent on each other for functional organization of proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein

P-glycoprotein (P-gp; ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The protein has two homologous halves joined by a linker region. Each half consists of a transmembrane (TM) domain with six TM segments and a nucleotide-binding domain. The drug substrate-binding pocket is at the interface between the TM segments in each half of the protein. Preliminary studies suggested that the arrangement of the two halves of P-gp shows rotational symmetry (i.e. "head-to-tail" arrangement). Here, we tested this model by determining whether the cytoplasmic ends of TM2 and TM3 in the N-terminal half are in close contact with TM11 in the C-terminal half. Mutants containing a pair of cysteines in TM2/TM11 or TM3/TM11 were subjected to oxidative cross-linking with copper phenanthroline. Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 degrees C, when thermal motion is reduced. Cross-linking was specific since no cross-linked product was detected in the 100 double Cys TM3/TM11 mutants. Vanadate trapping of nucleotide or the presence of some drug substrates inhibited cross-linking of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Cross-linking of TM2 and TM11 also blocked drug-stimulated ATPase activity. The close proximity of TM2/TM11 and TM5/TM8 (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692-7697) indicates that these regions between the two halves must enclose the drug-binding pocket at the cytoplasmic side of P-gp. They may form the "hinges" required for conformational changes during the transport cycle.     

Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 A resolution.

Electron cryomicroscopy of frozen-hydrated two-dimensional crystals of NhaA, a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembrane alpha-helices, has facilitated the calculation of a projection map of NhaA at 4.0 A resolution. NhaA was homologously expressed in E.coli with a His6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of approximately 38x48 A with a ring-shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22121 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter.     

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.     

Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.