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 Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor.

2021 ◽  
Author(s):  
Alessandro Barbieri ◽  
Nopnithi Thonghin ◽  
Talha Shafi ◽  
Stephen M Prince ◽  
Richard F Collins ◽  
...  
Keyword(s):  
The Body ◽  
Phase Plate ◽  
Binding Modes ◽  
Microscopy Imaging ◽  
Binding Domains ◽  
Atp Binding Cassette Transporter ◽  
Cryo Electron Microscopy ◽  
P Glycoprotein ◽  
A Site ◽  
Drug Free

ABCB1 (P-glycoprotein) is an ATP binding cassette transporter that is involved in the clearance of xenobiotics and it affects the disposition of many drugs in the body. Here we have studied ABCB1 in the drug-bound and drug-free states, simultaneously, using high contrast cryo-electron microscopy imaging and a Volta phase plate. The binding of the potent CFTR potentiator, ivacaftor, at a site in the central aqueous cavity is mediated by transmembrane α-helices 3,6,10,11 & 12. Binding is associated with a wider separation of the two halves of the transporter in the inward-facing state. Induced-fit changes the nucleotide binding domains in a way that may explain their increased affinity for ATP when drug is bound. Comparison of ivacaftor-bound structures of CFTR and ABCB1 suggests common features in the binding modes.

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.

ATP-binding Cassette Exporters: Structure and Mechanism with a Focus on P-glycoprotein and MRP1

2019 ◽  
Vol 26 (7) ◽  
pp. 1062-1078 ◽  
Author(s):  
Maite Rocío Arana ◽  
Guillermo Alejandro Altenberg
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Structural Information ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
And Function ◽  
Atp Binding Cassette

Background:Proteins that belong to the ATP-binding cassette superfamily include transporters that mediate the efflux of substrates from cells. Among these exporters, P-glycoprotein and MRP1 are involved in cancer multidrug resistance, protection from endo and xenobiotics, determination of drug pharmacokinetics, and the pathophysiology of a variety of disorders. Objective:To review the information available on ATP-binding cassette exporters, with a focus on Pglycoprotein, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Methods: Evaluation of selected publications on the structure and function of ATP-binding cassette proteins. Conclusions:Conformational changes on the nucleotide-binding domains side of the exporters switch the accessibility of the substrate-binding pocket between the inside and outside, which is coupled to substrate efflux. However, there is no agreement on the magnitude and nature of the changes at the nucleotide- binding domains side that drive the alternate-accessibility. Comparison of the structures of Pglycoprotein and MRP1 helps explain differences in substrate selectivity and the bases for polyspecificity. P-glycoprotein substrates are hydrophobic and/or weak bases, and polyspecificity is explained by a flexible hydrophobic multi-binding site that has a few acidic patches. MRP1 substrates are mostly organic acids, and its polyspecificity is due to a single bipartite binding site that is flexible and displays positive charge.

scholarly journals Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor

Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 923
Author(s):  
Alessandro Barbieri ◽  
Nopnithi Thonghin ◽  
Talha Shafi ◽  
Stephen M. Prince ◽  
Richard F. Collins ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Competitive Inhibitor ◽  
The Body ◽  
Atp Binding ◽  
Phase Plate ◽  
Microscopy Imaging ◽  
Binding Domains ◽  
Atp Binding Cassette Transporter ◽  
P Glycoprotein ◽  
Atp Binding Cassette

ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.

The P-glycoprotein multidrug transporter

2011 ◽  
Vol 50 ◽  
pp. 161-178 ◽  
Author(s):  
Frances J. Sharom
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Efflux Pump ◽  
Physiological Role ◽  
Binding Pocket ◽  
Binding Domains ◽  
Therapeutic Drugs ◽  
P Glycoprotein ◽  
Endogenous Metabolites ◽  
Outer Membrane Leaflet

Pgp (P-glycoprotein) (ABCB1) is an ATP-powered efflux pump which can transport hundreds of structurally unrelated hydrophobic amphipathic compounds, including therapeutic drugs, peptides and lipid-like compounds. This 170 kDa polypeptide plays a crucial physiological role in protecting tissues from toxic xenobiotics and endogenous metabolites, and also affects the uptake and distribution of many clinically important drugs. It forms a major component of the blood–brain barrier and restricts the uptake of drugs from the intestine. The protein is also expressed in many human cancers, where it probably contributes to resistance to chemotherapy treatment. Many chemical modulators have been identified that block the action of Pgp, and may have clinical applications in improving drug delivery and treating cancer. Pgp substrates are generally lipid-soluble, and partition into the membrane before the transporter expels them into the aqueous phase, much like a ‘hydrophobic vacuum cleaner’. The transporter may also act as a ‘flippase’, moving its substrates from the inner to the outer membrane leaflet. An X-ray crystal structure shows that drugs interact with Pgp within the transmembrane regions by fitting into a large flexible binding pocket, which can accommodate several substrate molecules simultaneously. The nucleotide-binding domains of Pgp appear to hydrolyse ATP in an alternating manner; however, it is still not clear whether transport is driven by ATP hydrolysis or ATP binding. Details of the steps involved in the drug-transport process, and how it is coupled to ATP hydrolysis, remain the object of intensive study.

scholarly journals Structural insight into substrate and inhibitor discrimination by human P-glycoprotein

Science ◽  
2019 ◽  
Vol 363 (6428) ◽  
pp. 753-756 ◽  
Author(s):  
Amer Alam ◽  
Julia Kowal ◽  
Eugenia Broude ◽  
Igor Roninson ◽  
Kaspar P. Locher
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Drug Binding ◽  
Therapeutic Drug ◽  
Binding Domains ◽  
Drug Binding Site ◽  
Cryo Electron Microscopy ◽  
P Glycoprotein ◽  
Insight Into

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo–electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate–hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

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.

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