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 journalArticle

Abstract

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
Volume279
Issue number33
Early online date10 Jun 2004
DOIs
Publication statusPublished - 13 Aug 2004

Fingerprint

P-Glycoprotein
Labeling
Topography
Cysteine
Hydrolysis
Nucleotides
Drug interactions
Pharmaceutical Preparations
Adenylyl Imidodiphosphate
Mutagenesis
Vanadates
Catalysis
Drug Interactions
Adenosine Diphosphate
Adenosine Triphosphatases
Adenosine Triphosphate
Binding Sites
Scanning
maleimide
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

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

Keywords

  • 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

Cite this

Rothnie, Alice ; Storm, Janet ; Campbell, Jeff ; Linton, Kenneth J. ; Kerr, Ian D. ; Callaghan, Richard. / The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 33. pp. 34913-34921.
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The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein. / Rothnie, Alice; Storm, Janet; Campbell, Jeff; Linton, Kenneth J.; Kerr, Ian D.; Callaghan, Richard.

In: Journal of Biological Chemistry, Vol. 279, No. 33, 13.08.2004, p. 34913-34921.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

AU - Rothnie, Alice

AU - Storm, Janet

AU - Campbell, Jeff

AU - Linton, Kenneth J.

AU - Kerr, Ian D.

AU - Callaghan, Richard

N1 - © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Publisher's version/PDF may be used after a 12 months embargo period

PY - 2004/8/13

Y1 - 2004/8/13

N2 - 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.

AB - 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.

KW - adenosine triphosphatases

KW - adenosine Triphosphate

KW - DNA sequence analysis

KW - binding sites

KW - boron compounds

KW - catalysis

KW - cell line

KW - cell membrane

KW - codon

KW - coumarins

KW - cysteine

KW - polyacrylamide gel electrophoresis

KW - hydrolysis

KW - insects

KW - kinetics

KW - Maleimides

KW - chemical models

KW - molecular models

KW - P-glycoprotein

KW - protein conformation

KW - protein isoforms

KW - tertiary protein structure

KW - recombinant proteins

UR - http://www.jbc.org/content/279/33/34913

U2 - 10.1074/jbc.M405336200

DO - 10.1074/jbc.M405336200

M3 - Article

VL - 279

SP - 34913

EP - 34921

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 33

ER -