Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site

Abstract

The human multidrug resistance P-glycoprotein uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. In this study, we used cysteine-scanning mutagenesis and cross-linking studies to identify residues that are exposed to the drug-binding site upon vanadate trapping. In the absence of nucleotides, C222(TM4) was cross-linked to C868(TM10) and C872(TM10); C306(TM5) was cross-linked to C868(TM10), C872(TM10), C945(TM11), C982(TM12), and C984(TM12); and C339(TM6) was cross-linked to C868(TM10), C872(TM10), C942(TM11), C982(TM12), and C985(TM12). These cysteines are in the middle of the predicted transmembrane (TM) segments and form the drug-binding site. Cross-linking between 332C(TM6) and cysteines introduced at the extracellular side of other TM segments was also done. In the absence of nucleotides, residues 332C and 856C on the extracellular side of TMs 6 and 10, respectively, were cross-linked with a 13-Å cross-linker (M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate). ATP plus vanadate inhibited cross-linking between 332C(TM6) and 856C(TM10) as well as those in the drug-binding site. Instead, vanadate trapping promoted cross-linking between 332C(TM6) and 976C(TM12) with a 10-Å cross-linker (M6M, 1,6-hexanediyl bismethanethiosulfonate). When ATP hydrolysis was allowed to proceed, then 332C(TM12) could form a disulfide bond with 975C(TM12). The cross-linking pattern of 332C(TM6) with residues in TM10 and TM12 indicates that the drug-binding site undergoes dynamic and relatively large conformational changes, and that different residues are exposed to the drug-binding site during the resting phase, upon vanadate trapping and at the completion of the catalytic cycle.T he human multidrug resistance P-glycoprotein (P-gp) is an ATP-dependent drug pump located at the plasma membrane that can transport a wide variety of structurally diverse compounds of different sizes (recently reviewed in ref. 1). P-gp is clinically important because overexpression of P-gp contributes to the phenomenon of multidrug resistance during treatment of AIDS and cancer. Many of these compounds in these treatment regimens are also substrates of P-gp (2-4). P-gp also plays an important role in mediating the bio-availability of oral drugs because of its relatively higher level of expression in the intestine, brain, liver, and kidney (5-10).P-gp consists of 1,280 aa that are organized in two repeating units of 610 aa that are joined by a linker region of about 60 aa (11). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (11-13).An important goal in understanding the mechanism of drug transport by P-gp is to determine how ATP hydrolysis is coupled to drug efflux. Both halves of P-gp can hydrolyze ATP (14, 15), but drug-stimulated ATPase activity (14) and conferring of drug resistance (16) requires interaction between the two halves of P-gp. Both halves of P-gp are required for activity because drug binding requires interaction between the NH 2 an...

Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11

“…A sample of the membrane was then incubated in the presence or absence of various concentrations of vinblastine, cyclosporin A, or rhodamine B for 15 min at 20°C. The samples were then cooled on ice for 10 min and treated with 0.2 mM concentrations of the homobifunctional methanethiosulfonate cross-linker 3,6,9,12-tetraoxatetradecane-1,14-diylbismethanethiosulfonate (M14M, 20.8 Å) (Toronto Research Chemicals, Toronto, ON) for 4 min on ice (9,22). The reactions were stopped by the addition of 2ϫ SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, and 4% (w/v) SDS) containing 50 mM EDTA and no reducing agent.…”

Introduction of the Most Common Cystic Fibrosis Mutation (ΔF508) into Human P-glycoprotein Disrupts Packing of the Transmembrane Segments

“…Cross-linking in the absence of vanadate was done at 37°C rather than 4°C because molecular motion might make it easier to form cross-links. Vanadate trapping of nucleotide locks the protein in the transition state (38) and can alter the conformational state of P-gp (39). For most of the mutants (e.g.…”

Identification and Characterization of the Binding Sites of P-Glycoprotein for Multidrug Resistance-Related Drugs and Modulators

“…In addition to this model of P-glycoprotein, another model has been proposed, which consists of two membrane-embedded sixteen-strand β-barrels, connected by short loops to two six-helix bundles beneath each barrel [45,46]. The involvement of TM segments and NBDs in substrate recognition and ATP binding/hydrolysis, respectively, have been established [47][48][49][50][51][52]. Substantial biochemical evidence, including changes in drug binding, epitope accessibility, fluorescent and spectroscopic measurements, and protease susceptibility [53][54][55][56][57][58][59], suggests that TM segments undergo conformational change upon nucleotide binding.…”

“…Considerable effort has been invested in characterizing drug resistance changes caused by Pglycoprotein mutations [52,53,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145]. P-glycoprotein mutations that have appeared in cultured cells selected for resistance to particular drugs [130][131][132], and mutations incorporated by site-directed mutagenesis have been evaluated [52,53,127,129,[134][135][136][137][138][139][140][141][142][143][144][145].…”