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Active transport of rhodamine 123 by the human multidrug transporter P-glycoprotein involves two independent outer gates.

ABSTRACT: Human P-glycoprotein (P-gp) is a multispecific drug-efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo-electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry-related outer gates. Experiments were aided by availability of the X-ray structure of a homodimeric eukaryotic homolog of P-gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation. We mutated analogous tyrosine residues in each half of the human full-length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry-related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry-related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P-gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates.

SUBMITTER: Nasim F

PROVIDER: S-EPMC7105846 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Active transport of rhodamine 123 by the human multidrug transporter P-glycoprotein involves two independent outer gates.

Nasim Fauzia F Schmid Diethart D Szakács Gergely G Sohail Azmat A Sitte Harald H HH Chiba Peter P Stockner Thomas T

Pharmacology research & perspectives 20200401 2

Human P-glycoprotein (P-gp) is a multispecific drug-efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo-electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry-related outer gates. Experiments were aided by availability of the X-ray structure of a homodimeric eukaryotic homolog of P-gp from red alga (CmABCB1), w ...[more]

PMID: 32232949

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Project description:P-glycoprotein (Pgp) is a major efflux pump in humans, overexpressed in a variety of cancers and associated with the development of multi-drug resistance. Allosteric modulation by various ligands (e.g., transport substrates, inhibitors, and ATP) has been biochemically shown to directly influence structural dynamics, and thereby, the function of Pgp. However, the molecular details of such effects, particularly with respect to the role and involvement of the surrounding lipids, are not well established. Here, we employ all-atom molecular dynamics (MD) simulations to study the conformational landscape of Pgp in the presence of a high-affinity, third-generation inhibitor, tariquidar, in comparison to the nucleotide-free (APO) and the ATP-bound states, in order to characterize the mechanical effects of the inhibitor that might be of relevance to its blocking mechanism of Pgp. Simulations in a multi-component lipid bilayer show a dynamic equilibrium between open(er) and more closed inward-facing (IF) conformations in the APO state, with binding of ATP shifting the equilibrium towards conformations more prone to ATP hydrolysis and subsequent events in the transport cycle. In the presence of the inhibitor bound to the drug-binding pocket within the transmembrane domain (TMD), Pgp samples more open IF conformations, and the nucleotide binding domains (NBDs) become highly dynamic. Interestingly, and reproduced in multiple independent simulations, the inhibitor is observed to facilitate recruitment of lipid molecules into the Pgp lumen through the two proposed drug-entry portals, where the lipid head groups from the cytoplasmic leaflet penetrate into and, in some cases, translocate inside the TMD, while the lipid tails remain extended into the bulk lipid environment. These "wedge" lipids likely enhance the inhibitor-induced conformational restriction of the TMD leading to the differential modulation of coupling pathways observed with the NBDs downstream. We suggest a novel inhibitory mechanism for tariquidar, and potentially for related third-generation Pgp inhibitors, where lipids are seen to enhance the inhibitory role in the catalytic cycle of membrane transporters.

Dataset Information

Active transport of rhodamine 123 by the human multidrug transporter P-glycoprotein involves two independent outer gates.

Publications

Active transport of rhodamine 123 by the human multidrug transporter P-glycoprotein involves two independent outer gates.

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