P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.

Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [ 3 H]25-hydroxycholesterol with an apparent K m of 0.7 M. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase mRNAs. Comparison of wild-type and ABCA1 Ϫ/Ϫ murine fibroblasts indicates that 25-hydroxycholesterol is effluxed ϳ5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1 Ϫ/Ϫ fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor. active transport; cholesterol homeostasis THE ATP BINDING CASSETTE (ABC) protein, ABCA1, was identified during a search for novel ABC proteins expressed in macrophages and at the time of its discovery, its function was unknown (34). Defects in the ABCA1 gene were subsequently shown to be the cause of Tangier disease, which is characterized by a lack, or abnormally low level, of high-density lipoprotein (HDL) and a markedly increased risk of coronary artery disease (5,7,11,56). Studies (2, 6, 14) using knockout mice have confirmed that ABCA1 plays a pivotal role in reverse cholesterol transport and in vitro, increased expression of the protein in several cell types results in elevated net efflux of cellular cholesterol and phospholipids. This efflux is dependent on the presence of an acceptor such as lipid-poor HDL or apolipoprotein A (apoA)-I (19, 70). Other proteins can also act as acceptors although their physiological relevance is presently unclear (46).The mechanism by which ABCA1 mediates efflux of cholesterol and phospholipids remains poorly defined. ABC transporters typically use the energy of ATP binding and hydrolysis to drive the transport of substrate across cellular membranes (26). In the case of hydrophobic compounds, transport is thought to involve ...

Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.

Structural changes in mouse P-glycoprotein (Pgp) induced by thermal unfolding were studied by differential scanning calorimetry (DSC), circular dichroism and fluorescence spectroscopy to gain insight into the solution conformation(s) of this ABC transporter that may not be apparent from current crystal structures. DSC of reconstituted Pgp showed two thermal unfolding transitions in the absence of MgATP, suggesting that each transition involved the cooperative unfolding of two or more interacting structural domains. A low calorimetric unfolding enthalpy and minimal structural changes were observed, which are hallmarks of the thermal unfolding of α-helical membrane proteins, because generally only the extramembranous regions undergo significant unfolding. Nucleotide binding increased the unfolding temperature of both transitions to the same extent, suggesting that one nucleotide binding domain (NBD) unfolds with each transition. Combined with the results from the two isolated NBDs, we propose that each DSC transition represents the cooperative unfolding of one NBD and the two contacting intracellular loops. Further, the presence of two transitions in both apo and MgATP bound wild-type Pgp suggests the NBD-dimeric conformation is transient, and that Pgp resides predominantly in the crystallographically observed inward-facing conformation with NBDs separated, even under conditions supporting continuous MgATP hydrolysis. In contrast, DSC of the vanadate-trapped MgADP·Pgp complex and the MgATP-bound catalytically inactive mutant, E552A/E1197A, show an additional transition at much higher temperature, corresponding to the unfolding of the nucleotide-trapped NBD-dimeric outward-facing conformation. The collective results indicate a strong preference for an NBD dissociated, inward-facing conformation of Pgp.