P-Glycoprotein Shows Strong Catalytic Cooperativity between the Two Nucleotide Sites

Senior, Alan E.; Bhagat, Sumedha

doi:10.1021/bi9719962

Cited by 107 publications

(75 citation statements)

References 29 publications

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“…The head-to-tail arrangement rationalizes the cooperativity in ATP hydrolysis that was observed in the maltose transporters and in human Mdr1 (Davidson et al, 1996;Senior and Bhagat, 1998). It is also consistent with earlier predictions based on sequence analysis (Jones and George, 1999) and with biochemical studies that demonstrate immediate vicinity of the P-loops and LSGGQ motifs Chen et al, 2001).…”

Section: Harnessing the Power Of Atp: Structure And Arrangement Of Thsupporting

confidence: 86%

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

Dawson

Hollenstein

Locher³

2007

Molecular Microbiology

140

123

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SummaryATP-binding cassette (ABC) transporters are integral membrane proteins that move diverse substrates across cellular membranes. ABC importers catalyse the uptake of essential nutrients from the environment, whereas ABC exporters facilitate the extrusion of various compounds, including drugs and antibiotics, from the cytoplasm. How ABC transporters couple ATP hydrolysis to the transport reaction has long remained unclear. The recent crystal structures of four complete ABC transporters suggest that a key step of the molecular mechanism is conserved in importers and exporters. Whereas binding of ATP promotes an outward-facing conformation, the release of the hydrolysis products ADP and phosphate promotes an inward-facing conformation. This basic scheme can in principle explain ATP-driven drug export and binding protein-dependent nutrient uptake.

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Section: Harnessing the Power Of Atp: Structure And Arrangement Of Thsupporting

confidence: 86%

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

Dawson

Hollenstein

Locher³

2007

Molecular Microbiology

140

123

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“…Previous data utilizing 8-azido-ATP labeling has indicated that both NBS are capable of binding nucleotide (12,13,29). Protection of both of the two NBS by ATP from NEM labeling or NBD-Cl labeling has also been noted, showing that ATP binds to both NBS (10,28,43). In addition both NBS have been shown to be catalytic sites (8).…”

Section: Discussionmentioning

confidence: 96%

“…Our laboratory has studied the two nucleotide sites, and we have established that both are catalytic MgATP hydrolysis sites, which interact together closely (7)(8)(9)(10). Earlier, we proposed (11) a catalytic mechanism for Pgp, in which the two NBS hydrolyze MgATP alternately, and hydrolysis is coupled to drug transport from an inner-facing, higher affinity, drug-binding site to an outer-facing, lower affinity site.…”

Section: P-glycoprotein (Pgp)mentioning

confidence: 98%

Large Scale Purification of Detergent-soluble P-glycoprotein fromPichia pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-type, Walker A, and Walker B Mutant Proteins

Lerner-Marmarosh¹,

Gimi²,

Urbatsch³

et al. 1999

Journal of Biological Chemistry

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145

213

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P-glycoprotein (Pgp; mouse MDR3) was expressed inPichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 Pglycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wildtype mouse MDR3 Pgp were studied using 2-(3)-O-(2,4,6-trinitrophenyl)adenosine tri-and diphosphate. Both analogs were found to bind with K d in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp. P-glycoprotein (Pgp)1 is a plasma membrane-located, ATPdriven drug efflux pump that confers multidrug resistance on mammalian cells (1-5). It occurs commonly in human tumors and is a major obstacle to successful chemotherapy. Consisting of two duplicated "halves" and a total length of around 1280 residues, it is a prominent member of the ABC transporter superfamily (6) and shows the typical ABC transporter domain arrangement consisting of two transmembrane domains (TMD) and two nucleotide-binding sites (NBS), in a linear sequence that can be represented as TMD1-NBS1-TMD2-NBS2. Current studies in many laboratories are aimed at understanding Pgp structure, function, normal physiology, and pharmacology.Our laboratory has studied the two nucleotide sites, and we have established that both are catalytic MgATP hydrolysis sites, which interact together closely (7-10). Earlier, we proposed (11) a catalytic mechanism for Pgp, in which the two NBS hydrolyze MgATP alternately, and hydrolysis is coupled to drug transport from an inner-facing, higher affinity, drug-binding site to an outer-facing, lower affinity site. Subsequent reports (12-16) have supported and extended this proposed mechanism. Further work (17-20) on the structure and location of the drug-binding sites has shown that certain transmembrane ␣-helices are involved in forming these sites, and work (21) with the closely related LmrA protein showed that transport is indeed from the inner lipid leaflet to the outer surface as proposed in Ref. 22. The mechanism of coupling of energy of ATP hydrolysis to transport of drugs is not well understood, however, at the present time. A preliminary structure of Pgp...

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“…Similarly, the ATPase activity of P-gp has previously been shown to be sensitive to the composition of the host lipid bilayer, and azidopine photolabeling experiments indicated that drug binding was not altered (35). Drugs are known to interact with the TMDs of P-gp (36)(37)(38), whereas ATP hydrolysis occurs in the cytosolic NBDs (39)(40)(41). A likely explanation for the increased potency of drugs to mediate the communication required to transmit drug binding events between the TMDs and NBDs in the sphingolipid/cholesterol-rich bilayer environment is an altered efficiency of allosteric conformational changes.…”

Section: The Kinetics Of [ 3 H]xr9576 Binding To P-gp Proteoliposomesmentioning

confidence: 99%

P-glycoprotein retains function when reconstituted into a sphingolipid- and cholesterol-rich environment

Modok

Heyward

Callaghan

2004

Journal of Lipid Research

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P-glycoprotein (P-gp) appears to be associated within specialized raftlike membrane microdomains. The activity of P-gp is sensitive to its lipid environment, and a functional association in raft microdomains will require that P-gp retains activity in the microenvironment. Purified hamster P-gp was reconstituted in liposomes comprising sphingomyelin and cholesterol, both highly enriched in membrane microdomains and known to impart a liquid-ordered phase to bilayers. The activity of P-gp was compared with that of proteoliposomes composed of crude egg phosphatidylcholine (unsaturated) or dipalmitoyl phosphatidylcholine (saturated) in the presence or absence of cholesterol. The maximal rate of ATP hydrolysis was not significantly altered by the nature of the lipid species. However, the potencies of nicardipine and XR9576 to modulate the ATPase activity of P-gp were increased in the sphingolipid-based proteoliposomes. The drug-P-gp interaction was investigated by measurement of the rates of The phenomenon of resistance to chemotherapy displayed by cancer cells is a significant clinical problem, and the underlying mechanisms are numerous. Resistance pathways are either inherent to the three-dimensional arrangement of the tissue or acquired after exposure to anticancer drugs. The plasma membrane plays a major role in resistance to chemotherapy by preventing the accumulation of drugs at efficacious concentrations. The principal mechanism is via active extrusion of anticancer drugs by transporters belonging to the ATP binding cassette (ABC) superfamily. The best-described and most widely observed ABC transporter in cancer tissues is P-glycoprotein (P-gp), whose expression is associated with a reduced rate of remission and poor prognosis. P-gp is an unusual transporter in that it recognizes an extremely broad range of drug substrates that are neither chemically nor functionally related. As a consequence, it is classified as a "multidrug" transporter, and the ability to recognize a range of drugs is attributable to the presence of multiple allosterically linked binding sites (1).The only common characteristic of drugs transported by P-gp is hydrophobicity, and several lines of evidence have demonstrated that the binding sites on the protein are in the intrabilayer region (2-5). Over the last 20 years, a consistent, if unexplained, observation has been that P-gp and its membrane environment display a complex and interwoven relationship [for review, see ref. (6)]. For example, the incorporation of P-gp into bilayers is known to alter lipid-packing properties (7), to produce ultrastructural changes in the membrane (8, 9), and has even been suggested to mediate the transbilayer translocation of specific phospholipids (7,10,11). More recent observations have proposed that P-gp is localized to detergentresistant low density membrane microdomains (12) and caveolae (13,14). In addition, the major lipid species (e.g., sphingolipids and cholesterol) found in caveolae and rafts display increased levels in several P-gp-contai...

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P-Glycoprotein Shows Strong Catalytic Cooperativity between the Two Nucleotide Sites

Cited by 107 publications

References 29 publications

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

Large Scale Purification of Detergent-soluble P-glycoprotein fromPichia pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-type, Walker A, and Walker B Mutant Proteins

P-glycoprotein retains function when reconstituted into a sphingolipid- and cholesterol-rich environment

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