Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

Abstract

Edited by Norma AllewellP-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-andclose motion alters the surface topology of P-gp within the drugbinding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.The association of multidrug resistance (MDR) 3 in cancer treatment with expression of human ABC transporters on the cell surface has raised the possibility that overcoming MDR might be achieved by inhibiting these transporters (1). One transporter in particular, the human P-glycoprotein (P-gp), has been well characterized; it is able to confer MDR by transporting numerous structurally unrelated drugs at the expense of hydrolyzing ATP (2, 3). In addition, P-gp plays important roles in drug distribution in normal physiology and is an essential component of many physiological barriers (4, 5).Long-standing efforts have been devoted to understanding the mechanism of P-gp function by various experimental approaches. Among them, structural studies of P-gp from various organisms have been reported (6 -8). In particular, a number of structures for the mouse P-gp (mP-gp) were obtained (8 -10). However, the diffraction limits of "near-native," inhibitor-bound, and nanobody-associated mP-gp crystals were relatively low, mostly near 4 Å resolution. The methylated protein gave higher resolution structures (11), but the impact of reductive methylation on the structure and function of P-gp was not addressed. The low resolution diffraction of P-gp crystals has been attributed in part to the intrinsic flexibility of the molecule (12-14).Mouse P-gp is a 1276-residue polypeptide and bears 87% sequence identity to human P-gp; it consists of two homologous halves connected by a flexible linker of ϳ75 residues. Each half is made of a transmembrane domain (TMD) implicated in drug recognition and transport and a nucleotide-binding domain (NBD). Each NBD is able to bind and hydrolyze ATP (15, 16), which is, however, dependent on the other NBD being functional (17,18). The two NBDs of P-gp are highly homologous, each featuring a consensus nucleotide-binding site with full-fledged Walker...

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale

“…This does not exclude any possible contribution of induced fit, and both may occur (25,26). Although induced fit has been suggested to be a source of promiscuity with P-gp (15,27), the likely contribution of conformational selection seems to have been ignored. In fact, the combination of induced fit and conformational selection may maximize substrate promiscuity of detoxication enzymes and transporters (28).…”

“…Another region that consistently shows a modest increase in deuterium exchange upon vanadate trapping is near the D and H loops of NBD2, but not observed in NBD1. In fact, this effect in NBD2 emphasizes the asymmetric effects of vanadate trapping that are well documented (15,17,23). Specifically, the Walker A (WA) peptide and the Q loop in NBD1 undergo a larger decrease in solvent accessibility with vanadate trapping compared to NBD2, and in general, NBD1 is more sensitive to ATP/vanadate than NBD2.…”

“…In addition, the transition state for ATP hydrolysis can be mimicked by addition of ATP, or ADP, and vanadate, which 'traps' a quasi-stable OF conformation (13,14). In contrast to Sav1886 or MsbA, however, recent data suggest that the IF to OF conformational transition of P-gp is less tightly coupled to nucleotide binding (15) or hydrolysis; even in the presence of excess nucleotide and vanadate, P-gp retains a substantial population of IF states with varying degrees of separation between NBDs (16,17). The emerging model suggests that drug-free P-gp samples a wide range of IF conformations that could bind different substrates or inhibitors, even with nucleotide bound.…”

Vasohibin 1 inhibits Adriamycin resistance in osteosarcoma cells via the protein kinase B signaling pathway

“…Drug resistance is an important characteristic of malignant tumors and has been an important factor in the failure of cancer treatment (15). ATP-binding cassette drug efflux pump P-gp has been proposed to serve crucial functions for tumor cells acquiring MDR (16,17). ADR is the first-line chemotherapy drug used to treat osteosarcoma.…”

Targeted delivery of ginsenoside compound K using TPGS/PEG-PCL mixed micelles for effective treatment of lung cancer

“…Once P-gp combines with drugs, the drug is pumped from inside the cell with energy provided by ATP, and the concentration of the drug in the cytoplasm decreases, resulting in a weak antitumor effect of the drug and removal of the drug from the cell. 22 TPGS could inhibit P-gp efflux by blocking ATP that provides energy for this activity, and thus, improve the antitumor effect of a drug.…”