title: Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein.
abstract: P-glycoprotein (P-gp; ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The protein has two homologous halves joined by a linker region. Each half consists of a transmembrane (TM) domain with six TM segments and a nucleotide-binding domain. The drug substrate-binding pocket is at the interface between the TM segments in each half of the protein. Preliminary studies suggested that the arrangement of the two halves of P-gp shows rotational symmetry (i.e. "head-to-tail" arrangement). Here, we tested this model by determining whether the cytoplasmic ends of TM2 and TM3 in the N-terminal half are in close contact with TM11 in the C-terminal half. Mutants containing a pair of cysteines in TM2/TM11 or TM3/TM11 were subjected to oxidative cross-linking with copper phenanthroline. Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 degrees C, when thermal motion is reduced. Cross-linking was specific since no cross-linked product was detected in the 100 double Cys TM3/TM11 mutants. Vanadate trapping of nucleotide or the presence of some drug substrates inhibited cross-linking of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Cross-linking of TM2 and TM11 also blocked drug-stimulated ATPase activity. The close proximity of TM2/TM11 and TM5/TM8 (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692-7697) indicates that these regions between the two halves must enclose the drug-binding pocket at the cytoplasmic side of P-gp. They may form the "hinges" required for conformational changes during the transport cycle.
authors.affiliation: Canadian Institutes of Health Research Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Tip W
authors.initials: TW
authors.lastname: Loo
authors.forename: M Claire
authors.initials: MC
authors.lastname: Bartlett
authors.forename: David M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids; D001120:Arginine; D001665:Binding Sites; D003545:Cysteine; D005998:Glycine; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D014633:Valine
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000596:Amino Acids; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; D014633:Valine; D003545:Cysteine; D005998:Glycine