title: Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains.
abstract: The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.
authors.affiliation: Canadian Institutes of Health Research Group in Membrane Biology, Departments of Medicine and Biochemistry, 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 / Q000737:chemistry; D001692:Biological Transport; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006801:Humans; D015151:Immunoblotting; D008954:Models, Biological; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D010649:Phenylalanine; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases