scholarly journals The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids

2005 ◽  
Vol 389 (2) ◽  
pp. 517-526 ◽  
Author(s):  
Paul D. W. Eckford ◽  
Frances J. Sharom
Keyword(s):  
Efflux Pump ◽  
Drug Binding ◽  
Sugar Residue ◽  
Multidrug Transporter ◽  
Membrane Phospholipids ◽  
Binding Domains ◽  
Hydrophobic Compounds ◽  
P Glycoprotein ◽  
Fluorescence Quenching Technique ◽  
Hydrolysis Of

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a ‘flippase’ for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD–GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD–GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD–GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD–lactosylceramide drastically reduced the rate of flipping compared with NBD–PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.

scholarly journals Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

2018 ◽  
Vol 115 (9) ◽  
pp. E1973-E1982 ◽  
Author(s):  
Amer Alam ◽  
Raphael Küng ◽  
Julia Kowal ◽  
Robert A. McLeod ◽  
Nina Tremp ◽  
...  
Keyword(s):  
Lipid Membrane ◽  
Binding Pocket ◽  
Conformational Epitope ◽  
Multidrug Transporter ◽  
Inhibitory Antibody ◽  
Binding Domains ◽  
Atp Binding Cassette Transporter ◽  
P Glycoprotein ◽  
Inhibitory Agents ◽  
Human Specific

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

scholarly journals Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

2020 ◽  
Vol 117 (47) ◽  
pp. 29609-29617
Author(s):  
Andaleeb Sajid ◽  
Sabrina Lusvarghi ◽  
Megumi Murakami ◽  
Eduardo E. Chufan ◽  
Biebele Abel ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cancer Drug ◽  
Binding Domains ◽  
Cancer Drug Resistance ◽  
P Glycoprotein ◽  
Cell Transforming

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

scholarly journals Allosteric Role of Substrate Occupancy Toward the Alignment of P-glycoprotein Nucleotide Binding Domains

2018 ◽  
Author(s):  
Lurong Pan ◽  
Stephen G Aller
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Lipid Bilayer ◽  
Small Molecule ◽  
Aromatic Amino Acids ◽  
Drug Binding ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Binding Cavity

AbstractP-glycoprotein (Pgp) is an ATP-binding cassette transporter that eliminates toxins from the cell but causes multidrug resistance in chemotherapies. The crystal structures of Pgp revealed drug-like compounds bound to an inward-facing conformation in which the energy-harnessing nucleotide binding domains (NBDs) were widely separated with no interfacial interaction. Following drug binding, inward-facing Pgp must transition to an NBD dimer conformation to achieve ATP binding and hydrolysis at canonical sites defined by both halves of the interface. However, given the high degree of flexibility shown for this transporter, it is difficult to envision how NBDs overcome entropic considerations for achieving proper alignment in order to form the canonical ATP binding site. We explored the hypothesis that substrate occupancy of the polyspecific drug-binding cavity plays a role in the proper alignment of NBDs using computational approaches. We conducted twelve atomistic molecular dynamics (MD) simulations (100-300 ns) on inward-facing Pgp in a lipid bilayer with and without small molecule substrates to ascertain effects of drug occupancy on NBD dimerization. Both apo- and drug-occupied simulations showed NBDs approaching each other compared to the crystal structures. Apo-Pgp reached a pseudo-dimerization in which NBD signature motifs for ATP binding exhibited a significant misalignment during closure. In contrast, occupancy of three established substrates positioned by molecular docking achieved NBD alignment that was much more compatible with a canonical NBD dimerization trajectory. Additionally, aromatic amino acids, known to confer the polyspecific drug-binding characteristic of the internal pocket, may also govern polyspecific drug access to the cavity. The enrichment of aromatics comprising the TM4-TM6 portal suggested a preferential pathway over the aromatic-poor TM10-TM12 for lateral drug entry from the lipid bilayer. Our study also suggested that drug polyspecificity is enhanced due to a synergism between multiple drug-domain interactions involving 36 residues identified in TM1, 5, 6, 7, 11 and 12.Author SummaryP-glycoprotein (Pgp) is an active drug pump known to cause clinical multi-drug resistance. The static atomic structure of Pgp was determined by trapping an inward-facing conformation bound to small molecule substrates by crystallization, however the effect of substrates on Pgp dynamics following binding is poorly understood. In this study, six apo-Pgp and six drug-occupied Pgp were simulated using unconstrained atomistic molecular dynamics (MD) for 100-300 ns. We demonstrate an allosteric communication of drug binding “from the top down”, that is from the TMDs to the NBDs that promotes NBD alignment and trajectories that favor canonical ATP binding. Other analyses suggested that aromatic amino acids in both the central drug-binding cavity and the “front portal” (TM4/TM6) confer polyspecific recognition. Additionally, comparison of the thermal B-factors between the experimental measurement and MD simulation indicated that different physical and chemical environments (temperature, in surfo vs. in meso, solution compositions) only alter the regional scales of thermal fluctuations but not the patterns of these motions. Lastly, DCCM and normal mode analyses were used to decipher thermal motions and the motion correlations between various domains in Pgp, allowing us to propose a substrate allosteric mechanism and an energy conservation mechanism during the catalytic cycle.

scholarly journals Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

2006 ◽  
Vol 401 (2) ◽  
pp. 597-605 ◽  
Author(s):  
Yasuhisa Kimura ◽  
Noriyuki Kioka ◽  
Hiroaki Kato ◽  
Michinori Matsuo ◽  
Kazumitsu Ueda
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Binding Site ◽  
Drug Binding ◽  
Soluble Form ◽  
Strongly Correlated ◽  
Drug Binding Site ◽  
Hydrophobic Compounds ◽  
P Glycoprotein ◽  
Human Mdr1

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug–MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on Km values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). Vmax values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

scholarly journals Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9Å resolution

2018 ◽  
Author(s):  
Nopnithi Thonghin ◽  
Richard F. Collins ◽  
Alessandro Barbieri ◽  
Talha Shafi ◽  
Alistair Siebert ◽  
...  
Keyword(s):  
Binding Sites ◽  
Drug Binding ◽  
Multi Drug Resistance ◽  
Binding Domains ◽  
Atp Binding Cassette Transporter ◽  
P Glycoprotein ◽  
Head Groups ◽  
Related Drug ◽  
Drug Binding Sites

AbstractP-glycoprotein (ABCB1) is a ATP-binding cassette transporter that plays an important role in the removal of drugs and xenobiotic compounds from the cell. It is also associated with multi-drug resistance in cancer. Here we report novel features of the cryo-EM-derived structure of P-glycoprotein in the post-hydrolytic state: The cytosolic nucleotide-binding domains (NBDs) are separated despite ADP remaining bound to the NBDs. Gaps in the TMDs that connect to the inner hydrophilic cavity are back-filled by detergent head-groups from the annular detergent micelle and are close to two regions predicted to delineate two pseudo-symmetry-related drug-binding sites. In this conformation, the (newly-resolved) N-terminal extension, NBD-TMD linker region and gap-filling detergents all appear to impede NBD dimerisation. We propose a model for the mechanism of action of the exporter where ATP will be bound to the protein for most of the time, consistent with the high physiological ATP concentrationsin vivo.

scholarly journals Studies of Human MDR1-MDR2 Chimeras Demonstrate the Functional Exchangeability of a Major Transmembrane Segment of the Multidrug Transporter and Phosphatidylcholine Flippase

1999 ◽  
Vol 19 (2) ◽  
pp. 1450-1459 ◽  
Author(s):  
Yi Zhou ◽  
Michael M. Gottesman ◽  
Ira Pastan
Keyword(s):  
Functional Relationship ◽  
Transmembrane Domain ◽  
Site Directed Mutagenesis ◽  
Multidrug Transporter ◽  
Transmembrane Segment ◽  
Glycoprotein P ◽  
Hydrophobic Compounds ◽  
Functional Differences ◽  
P Glycoprotein ◽  
Human Mdr1

ABSTRACT P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 andMDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).

scholarly journals P-glycoprotein Retains Drug-stimulated ATPase Activity upon Covalent Linkage of the Two Nucleotide Binding Domains at Their C-terminal Ends

2011 ◽  
Vol 286 (12) ◽  
pp. 10476-10482 ◽  
Author(s):  
Brandy Verhalen ◽  
Stephan Wilkens
Keyword(s):  
Atpase Activity ◽  
High Efficiency ◽  
Efflux Pump ◽  
Close Association ◽  
Nucleotide Binding ◽  
Cross Linking ◽  
Central Cavity ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Transporter Family

P-glycoprotein (Pgp), a member of the ABC transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anti-cancer chemotherapy. We have recently obtained EM projection images of lipid-bound Pgp without nucleotide and transport substrate that showed the two halves of the transporter separated by a central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125–40131). Addition of nucleotide and/or substrate lead to a close association of the two halves of the transporter, thereby closing the central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2008) J. Biol. Chem. 283, 5769–5779). Here, we used cysteine-mediated disulfide cross-linking to further delineate the structural rearrangements of the two nucleotide binding domains (NBD1 and NBD2) that take place during catalysis. Cysteines introduced at or near the C-terminal ends of NBD1 and NBD2 allowed for spontaneous disulfide cross-linking under nonreducing conditions. For mutant A627C/S1276C, disulfide formation was with high efficiency and cross-linked Pgp retained 30–68% drug-stimulated ATPase activity compared with reduced or cysteine-less Pgp. Two other cysteine pairs (K615C/S1276C and A627C/K1260C) also formed a disulfide but to a lesser extent, and the cross-linked form of these two mutants had lower drug-stimulated ATPase activity. The data suggest that the C-terminal ends of the two NBDs of Pgp are not required to undergo significant motion with respect to one another during the catalytic cycle.

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