scholarly journals Karanjin interferes with ABCB1, ABCC1, and ABCG2

2014 ◽  
Vol 17 (1) ◽  
pp. 92 ◽  
Author(s):  
Martin Michaelis ◽  
Florian Rothweiler ◽  
Thomas Nerreter ◽  
Mohsen Sharifi ◽  
Taravat Ghafourian ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Drug Transport ◽  
Multi Drug Resistance ◽  
Drug Efflux ◽  
Potential Drug ◽  
Antibacterial Effects ◽  
Low Concentrations ◽  
Substance Transport

PURPOSE: The prominent ATP-binding cassette (ABC) transporters ABCB1, ABCC1, and ABCG2 are involved in substance transport across physiological barriers and therefore in drug absorption, distribution, and elimination. They also mediate multi-drug resistance in cancer cells. Different flavonoids are known to interfere with different ABC transporters. Here, the effect of the furanoflavonol karanjin, a potential drug with antiglycaemic, gastroprotective, antifungal, and antibacterial effects, was investigated on ABCB1, ABCC1, and ABCG2-mediated drug transport in comparison to the flavonoids apigenin, genistein, and naringenin. METHODS: Cells expressing the relevant transporters (ABCB1: UKF-NB-3ABCB1, UKF-NB-3rVCR10; ABCC1: G62, PC-3rVCR20; ABCG2: UKF-NB-3ABCG2) were used in combination with specific fluorescent and cytotoxic ABC transporter substrates and ABC transporter inhibitors to study ABC transporter function. Moreover, the effects of the investigated flavonoids were determined on the ABC transporter ATPase activities. RESULTS: Karanjin interfered with drug efflux mediated by ABCB1, ABCC1, and ABCG2 and enhanced the ATPase activity of all three transporters. Moreover, karanjin exerted more pronounced effects than the control flavonoids apigenin, genistein, and naringenin on all three transporters. Most notably, karanjin interfered with ABCB1 at low concentrations being about 1µM. CONCLUSIONS: Taken together, these findings should be taken into account during further consideration of karanjin as a potential drug for different therapeutic indications. The effects on ABCB1, ABCC1, and ABCG2 may affect the pharmacokinetics of co-administered drugs. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.METHODS: Cells expressing the relevant transporters (ABCB1: UKF-NB-3ABCB1, UKF-NB-3rVCR10; ABCC1: G62, PC-3rVCR20; ABCG2: UKF-NB-3ABCG2) were used in combination with specific fluorescent and cytotoxic ABC transporter substrates and ABC transporter inhibitors to study ABC transporter function. Moreover, the effects of the investigated flavonoids were determined on the ABC transporter ATPase activities.RESULTS: Karanjin interfered with drug efflux mediated by ABCB1, ABCC1, and ABCG2 and enhanced the ATPase activity of all three transporters. Moreover, karanjin exerted more pronounced effects than the control flavonoids apigenin, genistein, and naringenin on all three transporters. Most notably, karanjin interfered with ABCB1 at low concentrations being about 1µM.CONCLUSIONS: Taken together, these findings should be taken into account during further consideration of karanjin as a potential drug for different therapeutic indications. The effects on ABCB1, ABCC1, and ABCG2 may affect the pharmacokinetics of co-administered drugs.

scholarly journals Transcriptional study of the ABC transporter-encoding genes in response to fungicide treatment and during plant infection in the phytopathogenic fungusBotrytis cinerea

2016 ◽  
Author(s):  
Elise Loisel ◽  
Isabelle R. Goncalves ◽  
Nathalie Poussereau ◽  
Marie-Claire Grosjean-Cournoyer ◽  
François Villalba ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Grey Mould ◽  
Multi Drug Resistance ◽  
Drug Efflux ◽  
Mould Fungus ◽  
Over Expression ◽  
Plant Infection ◽  
Commercially Important ◽  
Encoding Genes

AbstractThe grey mould fungusBotrytis cinereacauses worldwide losses of commercially important fruits, vegetables and ornamentals. Various fungicides, with different modes of action, are effective against this pathogen, but isolates with multiple fungicide-resistance phenotypes (Multi Drug Resistance, MDR) have been observed with increasing frequency. In fungi, ATP binding cassette (ABC) transporters participate in drug efflux and we report here on the parallel transcriptional study of the predicted ABC transporter-encoding genes inB. cinerea.During plant infection, transcription of all of these genes was activated and over-expression was observed for a few of these genes if the plants were treated with a fungicide prior to infection. In the absence of plant, most of the genes were transcriptionally activated in response to two different fungicides. Both common and specific transcriptional signatures were observed.

Erlotinib Antagonizes Efflux Via ABC Transporters and Decreases P-Gp Cell Surface Expression by Inhibiting SRC Kinase and mTOR Pathways in Acute Myeloid Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2564-2564 ◽  
Author(s):  
Elodie Lainey ◽  
Marie Sebert ◽  
Cyrielle Bouteloup ◽  
Carole Leroy ◽  
Sylvain Thepot ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Atpase Activity ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Hoechst 33342

Abstract Abstract 2564 Background: Erlotinib (Erlo) was originally developed as an epidermal growth factor receptor inhibitor, yet it also exerts antileukemic “off-target” effects, in vitro and in vivo in MDS and AML (Boehrer et al., Blood, 2008). In a preliminary pre-clinical study, we observed that Erlo increased chemosensitivity to current AML drugs in different AML cell lines and in ex vivo AML patient cells (n=3) (ASH 2010, 2163). Those first results suggested an implication of ABC-transporters in the potentiation of apoptosis. Here, we bring direct evidence for Erlo's ability to hinder efflux pumps and to decrease their expression on AML cells. Methods: Drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ or doxorubicin-Dox), and specific efflux via P-gp (substrates: DioC23 and Rho-123), MRP (s: Calcein and CDCFDA) and BCRP (s: Hoechst 33342) were quantified by FACS following incubation with 10mM Erlo. Intracellular VP-16) content was quantified by Rapid Resolution Liquid Chromatography (RRLC). Biochemical inhibitors of the respective ABC-transporters (CSA (1μM), verapamil (Vera-10μM), MK571 (10μM), KO143 (500nM) served as positive controls. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), Dox (100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assessment of sensitivity to the drug combinations listed above were carried out in KG-1 cells, and its more immature variant KG-1a and in ex vivo CD34+ marrow cells from AML patients (AML post MDS n=5, de novo AML n=5). P-gp's ATPase activity was quantified with the luminescence-based Pgp-Gloä Assay System. Surface expression of P-gp was determined by FACS analysis and total protein expression of MRP, BCRP and P-gp by immunoblot analysis. Functional relevance of signaling pathways was tested using the SRC inhibitor PP2 (10μM) and the mTOR inhibitor Rapamicin (10nM). Results: We found that I) Erlo inhibited efflux via P-gp, MRP and BCRP as demonstrated by increased intracellular retention of DioC23/Rho-123, Calcein/CDCFDA and Hoechst 33342, respectively, andby its ability to retain MTX (300nM) and Dox (200nM) intracellularly II) Inhibition of drug efflux was higher in KG-1 than in KG-1a cellss, in agreement with a lower expression of P-gp and BCRP on KG-1a as compared to KG-1 cells; III) Quantification of VP-16 by RRLC after incubation with or without Erlo showed the ability of Erlo to increase intracellular VP-16 contents by approximately 60%; IV) Erlo increased ATPase activity in a dose-dependant manner, supporting the notion that Erlo is a competitive inhibitor of P-gp; IV) Erlo combined to VP-16 induced synergistic effects on apoptosis in KG-1 cells, and to a lesser extent in KG-1a (48h KG-1: Erlo 20%, VP-16 38%, Erlo+VP16 78%, KG-1a 48h: Erlo 10%, VP-16: 12%, Erlo+VP16: 35%); V) 48h of incubation with Erlo reduced cell surface expression of P-gp in KG-1 cells by 50%, whereas total P-gp protein expression remained unchanged, suggesting that Erlo interferes exclusively with the protein form expressed on the cell surface, VI) Decrease of P-gp cell surface expression was recapitulated upon incubation with PP2 (10μM) or Rapamicin (10nM); VII) the combination of Erlo+VP-16 in 10 AML-patient samples induced synergistic effects on apoptosis in 5 of them and additive effects in 3 of them. Conclusions: We here confirm that Erlo increases sensitivity towards chemotherapeutic agents subjected to drug efflux via ABC-transporters and delineate the molecular pathways conveying these effects. Disclosures: Fenaux: Celgene: Honoraria, Research Funding.

scholarly journals Structural basis for the mechanism of ABC transporters

2015 ◽  
Vol 43 (5) ◽  
pp. 889-893 ◽  
Author(s):  
Konstantinos Beis
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Eukaryotic Cells ◽  
Structural Basis ◽  
Multi Drug Resistance ◽  
Hydrolysis Of ◽  
Transport Molecules ◽  
Alternating Access

The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

scholarly journals The Plasmodium falciparum protein PfMRP1 functions as an influx ABC transporter

2022 ◽  
Author(s):  
Miguel Silva ◽  
Carla Calçada ◽  
Nuno Osório ◽  
Vitória Baptista ◽  
Vandana Thathy ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Plasmodium Falciparum ◽  
Plasma Membrane ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Malaria Parasite ◽  
Drug Transport ◽  
Parasite Species ◽  
Antimalarial Activity ◽  
Falciparum Protein

Abstract Adenosine triphosphate (ATP)-binding cassette (ABC) transporters play an important role in mediating solute or drug transport across cellular membranes. Although this class of transporters has been well characterized in diverse organisms little is known about the physiological roles in Plasmodium falciparum, the deadliest malaria parasite species. We studied the Plasmodium falciparum Multidrug Resistance-associated Protein 1 (PfMRP1; PF3D7_0112200), an ABC transporter localized to the parasite plasma membrane, generating genetic disrupted parasites. We demonstrate that parasites with disrupted pfmrp1 are resistant to folate analogs, methotrexate and aminopterin, with antimalarial activity. This phenotype occurs due to reduction in compound accumulation in the parasite cytoplasm. Phylogenetic analysis supports pfmrp1 being distantly related to ABC transporters in other eukaryotes, suggesting an unusual function. We propose that PfMRP1 can act as a solute importer, a function not previously observed in this organism.

PK11195, a Peripheral Benzodiazepine Receptor (PBR) Ligand, Broadly Blocks Drug Efflux to Chemosensitize Leukemia and Myeloma Cells by a PBR-Independent, Direct Transporter-Modulating Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1533-1533
Author(s):  
Roland B. Walter ◽  
Jason Pirga ◽  
Michelle R. Cronk ◽  
Sasha J. Mayer ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multidrug Resistance ◽  
Atpase Activity ◽  
Cell Lines ◽  
Abc Transporter ◽  
Ectopic Expression ◽  
Benzodiazepine Receptor ◽  
Drug Efflux ◽  
Peripheral Benzodiazepine Receptor ◽  
Resistance Protein

Abstract Background: Multidrug resistance (MDR) is frequently associated with expression of anti-apoptotic proteins of the Bcl-2 family and/or ATP-binding cassette (ABC) transporter proteins. We previously showed that the peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux, establishing PK11195 as a promising MDR reversal agent. We have now assessed its effect on other ABC transporters, namely multidrug resistance protein (MRP) and breast cancer resistance protein (BCRP), and have explored the mechanism by which PK11195 blocks drug efflux, focusing on Pgp as a paradigm. Methods: Flow cytometry was used to measure efflux of the cytotoxic drug, mitoxantrone (MIT), that was inhibited by cyclosporine A (CSA; to assess Pgp function), MK-571 (to assess MRP function), or Ko143 or GF120918 (to assess BCRP function). MIT-induced toxicity was determined after 24 hours by flow cytometry with DiOC6(3) plus propidium iodide staining, or by 3H-thymidine incorporation. Hematopoietic cell lines were virally transduced with human pBR to assess the effect of pBR expression on PK11195 action. Specific 3H-PK11195 binding was determined by competition with 1000-fold excess unlabeled PK11195. Microsome and plasma membranes were prepared using sucrose gradients. ATPase activity was measured as the liberation of inorganic phosphate detected by absorbance at 800nM. Conformation-specific and conformation-insensitive anti-Pgp monoclonal antibodies were used to analyze PK11195 effects on Pgp by flow cytometry. Results: Using a panel of human acute myeloid leukemia and multiple myeloma cell lines with endogenous or ectopic expression of one or more ABC transporter proteins, and using primary leukemia cell samples, we found that PK11195 broadly inhibits ABC transporter function, affecting not only Pgp but also MRP and BCRP. PK11195 blocked efflux often more effectively than CSA in Pgp+, MRP+ and BCRP+ cells, blocked efflux as well as the MRP modulator, MK-571, in MRP+ cells, and inhibited as well or better than the BCRP modulator, GF120918, in BCRP+ cells. Compared to parental cell lines, sublines over-expressing Pgp, MRP, or BCRP showed relative MIT-resistance in cytotoxicity assays. PK11195 co-treatments significantly increased MIT cytotoxicity in such cell lines expressing relatively high levels of one or more clinically relevant drug efflux protein even when anti-apoptotic proteins were also expressed. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but that pBR expression is unnecessary for PK11195-mediated efflux inhibition. PK11195 bound plasma membrane sites in Pgp+ cells, stimulated Pgp-associated ATPase activity, and caused conformational changes of Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bound non-competitively in Pgp+ cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Discussion: PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism, and broadly blocks drug efflux by a pBR-independent, ABC transporter-dependent mechanism. Since PK11195 concentrations that are effective in vitro can be safely achieved in vivo, PK11195 may be a useful clinical chemosensitizer in patients with MDR+ malignancies.

scholarly journals Caffeic Acid Attenuates Multi-Drug Resistance in Cancer Cells by Inhibiting Efflux Function of Human P-Glycoprotein

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 247
Author(s):  
Yu-Ning Teng ◽  
Charles C.N. Wang ◽  
Wei-Chieh Liao ◽  
Yu-Hsuan Lan ◽  
Chin-Chuan Hung
Keyword(s):  
Atpase Activity ◽  
Caffeic Acid ◽  
Efflux Pump ◽  
Multi Drug Resistance ◽  
Drug Efflux ◽  
Promising Candidate ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Calcein Am ◽  
Drug Efflux Pump

Multidrug resistance (MDR) is a complicated ever-changing problem in cancer treatment, and P-glycoprotein (P-gp), a drug efflux pump, is regarded as the major cause. In the way of developing P-gp inhibitors, natural products such as phenolic acids have gotten a lot of attention recently. The aim of the present study was to investigate the modulating effects and mechanisms of caffeic acid on human P-gp, as well as the attenuating ability on cancer MDR. Calcein-AM, rhodamine123, and doxorubicin were used to analyze the interaction between caffeic acid and P-gp, and the ATPase activity of P-gp was evaluated as well. Resistance reversing effects were revealed by SRB and cell cycle assay. The results indicated that caffeic acid uncompetitively inhibited rhodamine123 efflux and competitively inhibited doxorubicin efflux. In terms of P-gp ATPase activity, caffeic acid exhibited stimulation in both basal and verapamil-stimulated activity. The combination of chemo drugs and caffeic acid resulted in decreased IC50 in ABCB1/Flp-InTM-293 and KB/VIN, indicating that the resistance was reversed. Results of molecular docking suggested that caffeic acid bound to P-gp through GLU74 and TRY117 residues. The present study demonstrated that caffeic acid is a promising candidate for P-gp inhibition and cancer MDR attenuation.

scholarly journals The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

2016 ◽  
Vol 60 (9) ◽  
pp. 5400-5411 ◽  
Author(s):  
Lea M. Hürlimann ◽  
Valentina Corradi ◽  
Michael Hohl ◽  
Guido V. Bloemberg ◽  
D. Peter Tieleman ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Abc Transporter ◽  
Drug Transport ◽  
Efflux Pumps ◽  
Drug Binding ◽  
Functional Coupling ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Content Type ◽  
Drug Efflux Pumps

ABSTRACTNosocomial infections withEnterococcus faecalisare an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters ofE. faecalisthat are annotated as drug efflux pumps. Deletion ofef0789-ef0790on the chromosome ofE. faecalisresulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context ofE. faecalis. In contrast, heterologous expression inLactococcus lactisrevealed that EfrAB, EfrCD, and the product ofef2226-ef2227(EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression inL. lactis. Since all seven transporters were purified as heterodimers after overexpression inL. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

scholarly journals ANTIBACTERIAL EFFECT OF FLAVONOIDS FROM PROPOLIS PRODUCED BY TRIGONA ON ATPASE ACTIVITY OF STREPTOCOCCUS MUTANS

2018 ◽  
Vol 9 ◽  
pp. 6
Author(s):  
Dian Erlianda ◽  
Mochamad Fahlevi Rizal ◽  
Sarworini B. Budiardjo
Keyword(s):  
Infectious Disease ◽  
Oral Health ◽  
Atpase Activity ◽  
Streptococcus Mutans ◽  
Antibacterial Effect ◽  
Antibacterial Effects ◽  
Basic Ingredient ◽  
Low Concentrations ◽  
Significant Difference

Objective: Caries are a major problem related to oral health among children. As it is an infectious disease, bacteria, especially Streptococcus mutans, play an important role in caries. Flavonoids have been known to have antibacterial effects, and ethanol, even at very low concentrations, reportedly has antibacterial effects and is widely used as a basic ingredient in drugs.Aim: This study aims to analyze the antibacterial effects of different concentrations of propolis flavonoids and ethanol against S. mutans ATPase activity.Methods: S. mutans cells were exposed to 0.5%, 0.25%, 0.1%, 0.075%, and 0.05% propolis flavonoids and then incubated in an anaerobic atmosphere for 48 h. The ATPase activity in these cells was then tested using a colorimetric assay.Results: No significant difference was found in the average ATPase activity of S. mutans after treatment with various concentrations of propolis flavonoids and ethanol.Conclusions: Thus, 0.5%, 0.25%, 0.1%, 0.075%, and 0.05% propolis flavonoids and 10% ethanol had similar effects against S. mutans ATPase activity. Further studies are necessary to evaluate the efficacy of flavonoids from Trigona spp. propolis. 

scholarly journals Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 201 ◽  
Author(s):  
Sergi Vaquer ◽  
Elisabet Cuyàs ◽  
Arnau Rabadán ◽  
Albert González ◽  
Felip Fenollosa ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Drug Transport ◽  
Structural Changes ◽  
Multi Drug Resistance ◽  
Engineering System ◽  
Analytical Sensitivity ◽  
Transport Activity ◽  
Simple Diffusion ◽  
Parabolic Flights ◽  
Gravitational Stress

Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay® (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and interesting research lines in biology and human physiology with the potential for significant benefits for both space and terrestrial medicine.

scholarly journals Chemo-mechanical Coupling in the Transport Cycle of a Type II ABC Transporter

2018 ◽  
Author(s):  
Koichi Tamura ◽  
Hiroshi Sugimoto ◽  
Yoshitsugu Shiro ◽  
Yuji Sugita
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Md Simulations ◽  
Coupling Mechanism ◽  
Transmembrane Helices ◽  
Mechanical Coupling ◽  
Binding Domains ◽  
Wide Range

AbstractAT P -binding cassette (ABC) transporters are integral membrane proteins that translocate a wide range of substrates across biological membranes, harnessing free energy from the binding and hydrolysis of ATP. To understand the mechanism of the inward- to outward-facing transition that could be achieved by tight regulation of ATPase activity through extensive conformational changes of the protein, we applied template-based iterative all-atom molecular dynamics (MD) simulation to the heme ABC transporter BhuUV-T. The simulations, together with biased MDs, predict two new conformations of the protein, namely, occluded (Occ) and outward-facing (OF) conformations. The comparison between the inward-facing crystal structure and the predicted two structures shows atomic details of the gating motions at the transmembrane helices and dimerization of the nucleotide-binding domains (NBDs). The MD simulations further reveal a novel role of the ABC signature motifs (LSGG[Q/E]) at the NBDs in decelerating ATPase activity in the Occ form through sporadic flipping of the side chains of the LSGG[Q/E] catalytic serine residues. The orientational changes are coupled to loose NBD dimerization in the Occ state, whereas they are blocked in the OF form where the NBDs are tightly dimerized. The chemo-mechanical coupling mechanism may apply to other types of ABC transporters having the conserved LSGG[Q/E] signature motifs.

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