The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Alice Rothnie, Janet Storm, Jeff Campbell, Kenneth J. Linton, Ian D. Kerr, Richard Callaghan

Research output: Contribution to journalArticlepeer-review


Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.
Original languageEnglish
Pages (from-to)34913-34921
Number of pages9
JournalJournal of Biological Chemistry
Issue number33
Early online date10 Jun 2004
Publication statusPublished - 13 Aug 2004

Bibliographical note

© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Publisher's version/PDF may be used after a 12 months embargo period


  • adenosine triphosphatases
  • adenosine Triphosphate
  • DNA sequence analysis
  • binding sites
  • boron compounds
  • catalysis
  • cell line
  • cell membrane
  • codon
  • coumarins
  • cysteine
  • polyacrylamide gel electrophoresis
  • hydrolysis
  • insects
  • kinetics
  • Maleimides
  • chemical models
  • molecular models
  • P-glycoprotein
  • protein conformation
  • protein isoforms
  • tertiary protein structure
  • recombinant proteins


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