Abstract
P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the occluded nucleotide-bound state.
Highlights
The multidrug transporter P-glycoprotein (Pgp, ABCB1) is a plasma membrane protein belonging to the ATP-binding cassette (ABC) superfamily which couples the efflux of a wide variety of chemically and structurally different compounds to the hydrolysis of ATP [1]
Used chemotherapy drugs are transported by Pgp, and its overexpression in tumour cells is linked to the multidrug resistant (MDR) phenotype that many human cancers present in the clinic [2,3]
Some puzzling features of the system still remain unexplained, including: cooperativity of ATP hydrolysis at low ATP concentrations; mixed inhibition of ATPase activity by Pi; the steep concentration dependence observed for Vi trapping with
Summary
The multidrug transporter P-glycoprotein (Pgp, ABCB1) is a plasma membrane protein belonging to the ABC superfamily which couples the efflux of a wide variety of chemically and structurally different compounds to the hydrolysis of ATP [1].Commonly used chemotherapy drugs are transported by Pgp, and its overexpression in tumour cells is linked to the multidrug resistant (MDR) phenotype that many human cancers present in the clinic [2,3]. The multidrug transporter P-glycoprotein (Pgp, ABCB1) is a plasma membrane protein belonging to the ABC superfamily which couples the efflux of a wide variety of chemically and structurally different compounds to the hydrolysis of ATP [1]. Following the first report of Pgp ATPase activity [4], studies characterizing ATP hydrolysis were conducted in the early 1990s with plasma membrane preparations from MDR celllines [5], partially purified [6,7] or purified detergent-solubilized. The catalytic cycle of the enzyme, its coupling to drug transport, and its inhibition by ortho-vanadate (Vi) have been studied by several research groups [12,13]. In 1995, Senior’s group published a minimal reaction pathway for hydrolysis of one molecule of ATP by Pgp, and Vi-induced inhibition of its catalytic activity [14]. The protein possesses two consensus sequences for ATP binding, located within the two provide new insights into the mode of action of Pgp, and some hypotheses about the nature of the occluded state
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