title: Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1.
abstract: Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.
authors.affiliation: Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, Arizona 85259, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhao
authors.forename: Xiu-Bao
authors.initials: XB
authors.lastname: Chang
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000225:Adenine / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D017997:Leukotriene C4 / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C505032:CFTR protein, human; D004279:DNA, Viral; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017997:Leukotriene C4; D014443:Tyrosine; D000255:Adenosine Triphosphate; D000225:Adenine; D003545:Cysteine