Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump

Abstract

Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent K D values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.mass spectrometry from native state | real time substrate monitoring P -glycoprotein (P-gp) is an ATP-driven low-specificity efflux pump that plays an important role in the clearance of xenotoxins (1, 2). P-gp is also a member of the ATP-Binding Cassette (ABC) family of transporters and exports hydrophobic cytotoxic compounds as well as natural products, cyclic, and linear peptides (1, 3-5). Overexpression of P-gp in tumor cells impairs targeted drug delivery and is a major pitfall for chemotherapies. Small molecule substrates partition in the plasma membrane (6, 7), before binding in the internal hydrophobic cavity formed in the inward conformation of the pump (8). Export is then thought to proceed in an ATP-dependent way through conformational changes from the inward to the outward facing forms, evidenced by FRET spectroscopy (9). Recent highresolution structures of eukaryotic P-gp from mouse (8) and Caenorhabditis elegans (10) were obtained in inward conformations. Two prokaryotic homologs of P-gp [Sav1866 (11) and MsbA (12)] were captured crystallographically in outward-facing conformers, reflecting ATP-bound states, as well as two different inward states for MsbA. From these X-ray structures it is possible to build up a picture of P-gp, alternating between inward-and outward-facing conformations.Despite decades of careful biochemical studies (13), and recent insights from crystallography, many questions remain, however. Specifically it has not yet been possible to trap P-gp in an outwardfacing state or to show how substrate binding activates ATPase act...

Molecular Basis for Structural Heterogeneity of an Intrinsically Disordered Protein Bound to a Partner by Combined ESI-IM-MS and Modeling

“…Previous studies on the N TAIL -P XD complex by small-angle X-ray scattering (SAXS) and NMR showed that the majority of N TAIL remains disordered in the bound state, thus supporting the fuzzy and heterogeneous nature of this complex [33,38,44]. Although hydrophobic complexes could be expected to dissociate in the gas phase, detection of such complexes by MS has been reported in some instances [45][46][47]. The results of this study show that the MeV N TAIL -P XD complex is amenable to MS analysis and that distinct conformational states can be detected.…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…This proof copy is the copyright property of the publisher and is confidential until formal publication. mass spectrometry as demonstrated by Marcoux et al (2013). They used purified mouse Abcb1 in a detergent (n-dodecyl-β-D-maltoside-DDM) containing buffer and analyzed direct lipid binding to Abcb1 and then determined the bound lipids by mass spectrometry.…”

“…They used purified mouse Abcb1 in a detergent (n-dodecyl-β-D-maltoside-DDM) containing buffer and analyzed direct lipid binding to Abcb1 and then determined the bound lipids by mass spectrometry. In this study, three cardiolipin molecules were found bound to the substrate-binding cavity of Abcb1, and by related calculations, the size and number of lipids present simultaneously in the substrate-binding pocket were also determined (Marcoux et al, 2013). As a further expansion of this study, Marcoux et al performed molecular docking in which they showed that cardiolipin-14 binds to K230 and K822 found in transmembrane helices TM4 and 9 previously shown to be involved in lipid sensing (see Section 4.1.1).…”

“…In a recent study, binding of drugs and cardiolipins with different sizes to the ABCB1 protein has been investigated by using mass spectrometry and molecular docking (Marcoux et al, 2013). Importantly, the computational and experimental results were concordant, and the presence of two molecules of the smaller cardiolipin CL14 or only one molecule of the larger CL24 was indicated in the internal binding pocket of ABCB1.…”

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

“…However, electron density in the low resolution electron cryo-microscopy single particle reconstruction envelope of TmrAB was assigned to the detergent micelle (9). In the low resolution crystal structure of the eukaryotic P-glycoprotein, no lipids were observed (10,11), but non-denaturing mass spectrometry identified tightly associated lipids and a clear dependence of ligand and lipid binding (12).…”