P-glycoprotein structure and evolutionary homologies

Analysis of multidrug resistant cell lines has led to the identification of the P-glycoprotein multigene family. Two of the three classes of mammalian P-glycoproteins have the ability to confer cellular resistance to a broad range of structurally and functionally diverse cytotoxic agents. P-glycoproteins are integral membrane glycoproteins comprised of two similar halves, each consisting of six membrane spanning domains followed by a cytoplasmic domain which includes a nucleotide binding fold. The P-glycoprotein is a member of a large superfamily of transport proteins which utilize ATP to translocate a wide range of substrates across biological membranes. This superfamily includes transport complexes comprised of multicomponent systems, half P-glycoproteins and P-glycoprotein-like homologs which appear to require approximately 12 α-helical transmembrane domains and two nucleotide binding folds for substrate transport. P-glycoprotein homologs have been isolated and characterized from a wide range of species. Amino acid sequences, the similarities between the halves and intron/exon boundaries have been compared to understand the evolutionary origins of the P-glycoprotein. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of Drosophila multidrug resistance gene homologs.

Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.     

Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily.

STE6, the yeast a-factor transporter, is a member of the ATP binding cassette protein superfamily, which also includes the mammalian multidrug resistance protein and the cystic fibrosis gene product. These proteins contain two homologous halves, each with six membrane spanning segments and a predicted ATP nucleotide binding domain. To assess the importance of the two halves of STE6, and to examine the functional significance of residues conserved among members of the ATP binding cassette superfamily, we introduced mutations into the nucleotide binding domains of STE6. Our analysis demonstrates that both halves of STE6 are critical for function and that some, but not all, mutations analogous to those known to result in cystic fibrosis impair STE6 activity. To examine further the functional contribution of each half of the STE6 protein, we severed the STE6 coding sequence and expressed the two halves of the transporter as separate polypeptides. Whereas 'half-molecules' are unable to provide transport function individually, co-expression of both half-molecules in the same cell leads to functional reconstitution of STE6-mediated a-factor transport.     

Haemonchus contortus: sequence heterogeneity of internucleotide binding domains from P-glycoproteins

P-Glycoproteins are transmembrane proteins associated with acquired multidrug resistance in mammalian cells and some protozoan parasites by a process of active drug export. P-glycoproteins contain two nucleotide binding domains which couple ATP to the drug transport process. The region between the nucleotide binding domains of P-glycoproteins, termed the internucleotide binding domain (IBD), was PCR-amplified from adult and larval cDNA libraries using degenerate primers. The 11 clones isolated by this method fall into several distinct groups, with one group of alleles displaying between 82 and 99% identity at the nucleotide level. This sets a baseline for sequence variation of transcribed alleles from a parasitic nematode. Northern blotting showed that P-glycoprotein genes are transcribed in a developmentally regulated fashion in Haemonchus contortus. Southern blots of H. contortus drug-resistant isolates with an IBD probe revealed a pattern consistent with the involvement of P-glycoprotein in resistance to avermectin/milbemycin anthelmintics.     

Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA

Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme–substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme–product complex (trapped by ADP-vanadate). 20 Å resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.     

A P-glycoprotein protects Caenorhabditis elegans against natural toxins.

P-glycoproteins can cause resistance of mammalian tumor cells to chemotherapeutic drugs. They belong to an evolutionarily well-conserved family of ATP binding membrane transporters. Four P-glycoprotein gene homologs have been found in the nematode Caenorhabditis elegans; this report describes the functional analysis of two. We found that PGP-3 is expressed in both the apical membrane of the excretory cell and in the apical membrane of intestinal cells, whereas PGP-1 is expressed only in the apical membrane of the intestinal cells and the intestinal valve. By transposon-mediated deletion mutagenesis we generated nematode strains with deleted P-glycoprotein genes and found that the pgp-3 deletion mutant, but not the pgp-1 mutant, is sensitive to both colchicine and chloroquine. Our results suggest that soil nematodes have P-glycoproteins to protect themselves against toxic compounds made by plants and microbes in the rhizosphere.     

Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural and functional characteristics, this bacterial protein is classified as a member of the P-glycoprotein cluster of the ABC transporter superfamily. LmrA can even substitute for P-glycoprotein in human lung fibroblast cells, suggesting that this type of transporter is conserved from bacteria to man. The functional similarity between bacterial LmrA and human P-glycoprotein is further exemplified by their currently known spectrum of substrates, consisting mainly of hydrophobic cationic compounds. In addition, LmrA was found to confer resistance to eight classes of broad-spectrum antibiotics, and homologs of LmrA have been found in pathogenic bacteria, supporting the clinical and academic value of studying this bacterial protein. Current studies are focused on unraveling the mechanism by which ABC multidrug transporters, such as LmrA, couple the hydrolysis of ATP to the translocation of drugs across the membrane. Recent evidence indicates that LmrA mediates drug transport by an alternating two-site transport mechanism.     

Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity

P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.     

Characterization of the azidopine and vinblastine binding site of P-glycoprotein.

To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.     

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.     

Spectroscopic and biophysical approaches for studying the structure and function of the P-glycoprotein multidrug transporter.

Multidrug resistance is a serious obstacle to the successful chemotherapeutic treatment of many human cancers. A major cause of multidrug resistance is the overexpression of a 170-kDa plasma membrane protein, known as P-glycoprotein, which appears to function as an ATP-driven efflux pump with a very broad specificity for hydrophobic drugs, peptides, and natural products. P-Glycoprotein is a member of the ABC superfamily and is proposed to consist of two homologous halves, each comprising six membrane-spanning segments and a cytosolic nucleotide binding domain. In recent years, P-glycoprotein has been purified and functionally reconstituted into lipid bilayers, where it retains both ATPase and drug transport activity. The availability of purified active protein has led to substantial advances in our understanding of the molecular structure and mechanism of action of this unique transporter. This review will focus on the recent application of fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, electron microscopy, and other biophysical techniques to the study of P-glycoprotein structure and function.     

Molecular analysis of the multidrug transporter, P-glycoprotein

Inherent or acquired resistance of tumor cells to cytotoxic drugs represents a major limitation to the successful chemotherapeutic treatment of cancer. During the past three decades dramatic progress has been made in the understanding of the molecular basis of this phenomenon. Analyses of drug-selected tumor cells which exhibit simultaneous resistance to structurally unrelated anti-cancer drugs have led to the discovery of the human MDR1 gene product, P-glycoprotein, as one of the mechanisms responsible for multidrug resistance. Overexpression of this 170 kDa N-glycosylated plasma membrane protein in mammalian cells has been associated with ATP-dependent reduced drug accumulation, suggesting that P-glycoprotein may act as an energy-dependent drug efflux pump. P-glycoprotein consists of two highly homologous halves each of which contains a transmembrane domain and an ATP binding fold. This overall architecture is characteristic for members of the ATP-binding cassette or ABC superfamily of transporters. Cell biological, molecular genetic and biochemical approaches have been used for structure-function studies of P-glycoprotein and analysis of its mechanism of action. This review summarizes the current status of knowledge on the domain organization, topology and higher order structure of P-glycoprotein, the location of drug- and ATP binding sites within P-glycoprotein, its ATPase and drug transport activities, its possible functions as an ion channel, ATP channel and lipid transporter, its potential role in cholesterol biosynthesis, and the effects of phosphorylation on P-glycoprotein activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance.

A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional drug therapies. This phenomenon is due in large part to the overexpression of a 170 kDa plasma membrane ATP-dependent pump known as the multidrug resistance transporter or P-glycoprotein. P-glycoprotein is a member of the large ATP binding cassette (ABC) superfamily of membrane transporters. This review focuses on the use of structure-function analyses to elucidate further the mechanism of action of mammalian P-glycoproteins. Ultimately, a complete understanding of the mechanism is important for the development of novel strategies for the treatment of many human cancers.     

Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.     

ABC transporters in lipid transport

Since it was found that the P-glycoproteins encoded by the MDR3 (MDR2) gene in humans and the Mdr2 gene in mice are primarily phosphatidylcholine translocators, there has been increasing interest in the possibility that other ATP binding cassette (ABC) transporters are involved in lipid transport. The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids. Both transporters can transport a wide range of hydrophobic drugs and may see lipid analogues as just another drug. The MDR3 gene probably arose in evolution from a drug-transporting P-glycoprotein gene. Recent work has shown that the phosphatidylcholine translocator has retained significant drug transport activity and that this transport is inhibited by inhibitors of drug-transporting P-glycoproteins. Whether the phosphatidylcholine translocator also functions as a transporter of some drugs in vivo remains to be seen. Three other ABC transporters were recently shown to be involved in lipid transport: ABCR, also called Rim protein, was shown to be defective in Stargardt's macular dystrophy; this protein probably transports a complex of retinaldehyde and phosphatidylethanolamine in the retina of the eye. ABC1 was shown to be essential for the exit of cholesterol from cells and is probably a cholesterol transporter. A third example, the ABC transporter involved in the import of long-chain fatty acids into peroxisomes, is discussed in the chapter by Hettema and Tabak in this volume.     

Heterologous expression systems for P-glycoprotein:E. coli,yeast, and baculovirus

Chemotherapy, though it remains one of the front-line weapons used to treat human cancer, is often ineffective due to drug resistance mechanisms manifest in tumor cells. One common pattern of drug resistance, characterized by simultaneous resistance to multiple amphipathic, but otherwise structurally dissimilar anticancer drugs, is termed multidrug resistance. Multidrug resistance in various model systems, covering the phylogenetic range from bacteria to man, can be conferred by mammalian P-glycoproteins (PGPs), often termed multidrug transporters. PGPs are 170-kD polytopic membrane proteins, predicted to consist of two homologous halves, each with six membrane spanning regions and one ATP binding site. They are members of the ATP-binding cassette (ABC) superfamily of transporters, and are known to function biochemically as energy-dependent drug efflux pumps. However, much remains to be learned about PGP structure-function relationships, membrane topology, posttranslational regulation, and bioenergetics of drug transport. Much of the recent progress in the study of the human and mouse PGPs has come from heterologous expression systems which offer the benefits of ease of genetic selection and manipulation, and/or short generation times of the organism in which PGPs are expressed, and/or high-level expression of recombinant PGP. Here we review recent studies of PGP inE. coli, baculovirus, and yeast systems and evaluate their utility for the study of PGPs, as well as other higher eukaryotic membrane proteins.     

Sequence of mdr3 cDNA encoding a human P-glycoprotein

We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.     

Molecular analysis of the multidrug transporter

The multidrug resistance gene product, P-glycoprotein or the multidrug transporter, confers multidrug resistance to cancer cells by maintaining intracellular levels of cytotoxic agents below a killing threshold. P-glycoprotein is located within the plasma membrane and is thought to act as an energy-dependent drug efflux pump. The multidrug transporter represents a member of the ATP-binding cassette superfamily of transporters (or traffic ATPases) and is composed of two highly homologous halves, each of which harbors a hydrophobic transmembrane domain and a hydrophilic ATP-binding fold. This review focuses on various biochemical and molecular genetic approaches used to analyze the structure, function, and mechanism of action of the multidrug transporter, whose most intriguing feature is its ability to interact with a large number of structurally and functionally different amphiphilic compounds. These studies have underscored the complexity of this membrane protein which has recently been suggested to assume alternative topological and quaternary structures, and to serve multiple functions both as a transporter and as a channel. With respect to the multidrug transporter activity of P-glycoprotein, progress has been made towards the elucidation of essential amino acid residues and/or polypeptide regions. Furthermore, the drug-stimulatable ATPase activity of P-glycoprotein has been established. The mechanism of drug transport by P-glycoprotein, however, is still unknown and its physiological role remains a matter of speculation.     

Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport.

P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.     

Stoichiometry and affinity of nucleotide binding to P-glycoprotein during the catalytic cycle

Drug transport mediated by P-glycoprotein (Pgp) is driven by hydrolysis of ATP at the two cytosolic nucleotide binding domains. However, little is currently known concerning the stoichiometry of nucleotide binding and how both stoichiometry and binding affinity change during the catalytic cycle of the transporter. To address this issue, we used fluorescence techniques to measure both the number of nucleotides bound to P-glycoprotein during various stages of the catalytic cycle and the affinity of nucleotide binding. Results showed that resting state P-glycoprotein bound two molecules of the fluorescent nucleotide derivative, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), whereas the vanadate-trapped transition state bound only one nucleotide molecule. Both resting and transition state P-glycoprotein showed similar affinity for TNP-ATP/TNP-ADP and unlabeled ATP/ADP. Following binding of various drugs, resting state P-glycoprotein displayed a higher affinity for nucleotides, up to 4-fold depending on the compound used. In contrast, the transition state showed substantially lower (up to 3-fold) nucleotide binding affinity when the drug binding site(s) is/are occupied. These results indicate that both nucleotide binding domains of P-glycoprotein are likely to be occupied with either ATP (or ADP) in the resting state and the transition state in the absence of transport substrates. Drugs alter the binding affinity to favor association of ATP with P-glycoprotein at the start of the catalytic cycle and release of ADP from the transition state following nucleotide hydrolysis.