Long Range Communication between the Drug-Binding Sites and Nucleotide Binding Domains of the Efflux Transporter ABCB1

Abstract

The ABC efflux pump P-glycoprotein (P-gp) transports a wide variety of drugs and is inhibited by others. Some drugs stimulate ATP hydrolysis at the nucleotide binding domains (NBDs) and are transported, others uncouple ATP hydrolysis and transport, and others inhibit ATP hydrolysis. The molecular basis for the different behavior of these drugs is not well understood despite the availability of several structural models of P-gp complexes with ligands bound. Hypothetically, ligands differentially alter the conformational dynamics of peptide segments that mediate the coupling between the drug binding sites and the NBDs. Here, we explore by hydrogen-deuterium exchange mass spectrometry the dynamic consequences of a classic substrate and inhibitor, vinblastine and zosuquidar, binding to mouse P-gp (mdr1a) in lipid nanodiscs. The dynamics of P-gp in nucleotide-free, pre-hydrolysis, and post-hydrolysis states in the presence of each drug reveal distinct mechanisms of ATPase stimulation and implications for transport. For both drugs, there are common regions affected in a similar manner, suggesting that particular networks are the key to stimulating ATP hydrolysis. However, drug binding effects diverge in the post-hydrolysis state, particularly in the intracellular helices (ICHs 3 and 4) and neighboring transmembrane helices. The local dynamics and conformational equilibria in this region are critical for the coupling of drug binding and ATP hydrolysis and are differentially modulated in the catalytic cycle.

Ligand’s Partition to the Lipid Bilayer Should Be Accounted for When Estimating Their Affinity to Proteins

“…The binding of ligands to P-gp has been characterized in their native membranes, [ 15 , 16 , 17 , 84 , 85 , 86 , 87 ] and with the purified protein solubilized in detergent micelles [ 17 ], reconstituted in nanodiscs [ 17 , 88 , 89 , 90 ] or proteoliposomes of different lipid composition [ 17 , 91 , 92 , 93 , 94 , 95 , 96 ]. The total concentration of protein varies from 2.5 μg/mL (0.025% w / v ) for purified P-gp reconstituted in liposomes to 1 mg/mL (0.1% w / v ) when using native membranes of cells overexpressing P-gp.…”

Hydrogen/deuterium exchange-mass spectrometry of integral membrane proteins in native-like environments: current scenario and the way forward

“…The group of William Atkins performed extensive HDX-MS studies on nanodisc-reconstituted P-gp [30][31][32], an ATP-binding cassette (ABC) transporter involved in the efflux of xenobiotics. In their initial study, they compared the conformational dynamics of P-gp in the ligand-free state vs the ADP-trapped state induced by vanadate.…”

“…Moreover, some may affect the coupling between these two domains. To understand the molecular mechanism underlying this difference, comparisons were drawn between the effect of substrate vinblastine and the transport inhibitor zosuquidar on the nucleotide free, prehydrolytic and posthydrolytic states of P-gp in DMPC nanodiscs [ 32 ]. A major difference was seen in the zosuquidar incubated posthydrolytic state where an overall deprotection was observed at the TMDs and NBDs.…”

Multidrug Resistance of Cancer Cells and the Vital Role of P-Glycoprotein

“…Both ATP-binding domains are found on the cytoplasmic intracellular part of the protein. In 2009, the first high-resolution X-ray crystallography structure of mouse P-gp was reported, with 87% homology to human P-gp [35,36]. Further investigation at a resolution up to 3.3 revealed primarily comparable tertiary structural features.…”