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

Julien Marcoux, Sheila C Wang, Argyris Politis, Eamonn Reading, Jerome Ma, Philip C Biggin, Min Zhou, Houchao Tao, Qinghai Zhang, Geoffrey Chang, Nina Morgner, Carol V Robinson

Research output: Contribution to journalArticlepeer-review

147 Citations (Scopus)
22 Downloads (Pure)

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 KD 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.

Original languageEnglish
Pages (from-to)9704-9
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number24
DOIs
Publication statusPublished - 11 Jun 2013

Keywords

  • ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry
  • Binding, Competitive
  • Biological Transport
  • Cardiolipins/chemistry
  • Cluster Analysis
  • Cyclosporine/chemistry
  • Detergents/chemistry
  • Drug Resistance, Multiple
  • Humans
  • Immunosuppressive Agents/chemistry
  • Kinetics
  • Lipids/chemistry
  • Mass Spectrometry/methods
  • Models, Molecular
  • Molecular Conformation
  • Nucleotides/chemistry
  • Pharmaceutical Preparations/chemistry
  • Protein Binding
  • Protein Conformation

Fingerprint

Dive into the research topics of 'Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump'. Together they form a unique fingerprint.

Cite this