scholarly journals Three-dimensional Structures of the Mammalian Multidrug Resistance P-glycoprotein Demonstrate Major Conformational Changes in the Transmembrane Domains upon Nucleotide Binding

2002 ◽  
Vol 278 (10) ◽  
pp. 8294-8299 ◽  
Author(s):  
Mark F. Rosenberg ◽  
Alhaji Bukar Kamis ◽  
Richard Callaghan ◽  
Christopher F. Higgins ◽  
Robert C. Ford
Keyword(s):  
Multidrug Resistance ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Nucleotide Binding ◽  
Transmembrane Domains ◽  
P Glycoprotein

scholarly journals Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein

2001 ◽  
Vol 276 (15) ◽  
pp. 11653-11661 ◽  
Author(s):  
Zuben E. Sauna ◽  
Suresh V. Ambudkar
Keyword(s):  
Multidrug Resistance ◽  
Functional Outcomes ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Catalytic Cycle ◽  
Nucleotide Binding Sites ◽  
P Glycoprotein ◽  
Random Manner

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis à visthe substrate, they show comparablet12for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

scholarly journals Screening of Natural Compounds as P-Glycoprotein Inhibitors against Multidrug Resistance

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 357
Author(s):  
Sérgio M. Marques ◽  
Lucie Šupolíková ◽  
Lenka Molčanová ◽  
Karel Šmejkal ◽  
David Bednar ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Cancer Drug ◽  
Binding Modes ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Anti Cancer ◽  
Theoretical Predictions ◽  
Glycoprotein Inhibitors

Multidrug resistance (MDR) is a common problem when fighting cancer with chemotherapy. P-glycoprotein (P-gp, or MDR1) is an active pump responsible for the efflux of xenobiotics out of the cell, including anti-cancer drugs. It is a validated target against MDR. No crystal structure of the human P-gp is available to date, and only recently several cryo-EM structures have been solved. In this paper, we present a comprehensive computational approach that includes constructing the full-length three-dimensional structure of the human P-gp and its refinement using molecular dynamics. We assessed its flexibility and conformational diversity, compiling a dynamical ensemble that was used to dock a set of lignan compounds, previously reported as active P-gp inhibitors, and disclose their binding modes. Based on the statistical analysis of the docking results, we selected a system for performing the structure-based virtual screening of new potential P-gp inhibitors. We tested the method on a library of 87 natural flavonoids described in the literature, and 10 of those were experimentally assayed. The results reproduced the theoretical predictions only partially due to various possible factors. However, at least two of the predicted natural flavonoids were demonstrated to be effective P-gp inhibitors. They were able to increase the accumulation of doxorubicin inside the human promyelocytic leukemia HL60/MDR cells overexpressing P-gp and potentiate the antiproliferative activity of this anti-cancer drug.

scholarly journals Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter

2006 ◽  
Vol 399 (2) ◽  
pp. 315-323 ◽  
Author(s):  
Paula L. Russell ◽  
Frances J. Sharom
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Functional Characterization ◽  
Tryptophan Fluorescence ◽  
Fold Increase ◽  
Nucleotide Binding ◽  
Forward Direction ◽  
Multidrug Transporter ◽  
Bivalent Cation ◽  
P Glycoprotein

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (Vi) and fluoroaluminate (AlFx) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp–M2+–ADP–X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeFx) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and Vi, BeFx or AlFx using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with Vi and ATP led to a 6-fold increase in the acrylamide quenching constant, KSV, suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with Vi and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)–ATP/TNP–ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeFx-trapped complex was much more polar than that of the Vi and AlFx complexes. Functionally, all the trapped complexes were able to bind drugs and TNP–nucleotides with unchanged affinity compared with native Pgp.

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.

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