Detergents as intrinsic P-glycoprotein substrates and inhibitors

Li-Blatter, Xiaochun; Nervi, Pierluigi; Seelig, Anna

doi:10.1016/j.bbamem.2009.07.010

Cited by 63 publications

(67 citation statements)

References 41 publications

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“…This could be one of the reasons for the modest changes in K d that were observed in these experiments. Another reason might be interferences from the detergent itself, which may also bind as a substrate (30,31) (14). Significant hydrolysis of TNP-ATP in this assay was prevented by the exclusion of Mg 2ϩ from the assay buffer.…”

Section: Resultsmentioning

confidence: 99%

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

Doshi

Veen

2013

Journal of Biological Chemistry

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Background: During substrate transport, ATP-binding cassette exporters switch between an inward-facing and an outward-facing state in a nucleotide-dependent fashion. Results: Substrate binding to bacterial MsbA initiates dimerization of nucleotide-binding domains without opening the membrane domains at their external side. Conclusion: Substrate binding to MsbA stabilizes an "inward-facing closed" pre-translocation state that binds ATP. Significance: Our observations suggest a fundamental mechanism by which substrates stimulate ATP hydrolysis.

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Section: Resultsmentioning

confidence: 99%

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

Doshi

Veen

2013

Journal of Biological Chemistry

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“…transport detergent as substrates and with the X-ray structure of P-gp from C. elegans wherein two molecules of n-undecyl-β-maltoside (UDM) were located inside the cavity (10,15).…”

Section: Resultsmentioning

confidence: 99%

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

Marcoux¹,

Wang²,

Politis³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

163

151

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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...

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“…Strong ionic detergents, such as SDS, may irreversibly destroy transporter function. Still, some nonionic detergents, such as Triton X-100 or C12EO8, below their CMC concentrations, have been indicated to be transported substrates of ABCB1 and possibly bind to the drug-binding site of the protein (Beck et al, 2013;Li-Blatter, Nervi, & Seelig, 2009;Seelig & Gerebtzoff, 2006;Zordan-Nudo, Ling, Liu, & Georges, 1993). Bile acids and related compounds (e.g., CHAPS) modify ABCG2 transporter function below the concentrations causing detergent effects, while some potentially "mild" detergents may have serious adverse effects.…”

Section: Article In Pressmentioning

confidence: 99%