title: The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette.
abstract: Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP).
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: In-Wha
authors.initials: IW
authors.lastname: Kim
authors.forename: Xiang-Hong
authors.initials: XH
authors.lastname: Peng
authors.forename: Zuben E
authors.initials: ZE
authors.lastname: Sauna
authors.forename: Peter C
authors.initials: PC
authors.lastname: FitzGerald
authors.forename: Di
authors.initials: D
authors.lastname: Xia
authors.forename: Marianna
authors.initials: M
authors.lastname: Müller
authors.forename: Krishnamachary
authors.initials: K
authors.lastname: Nandigama
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000032:analysis; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D017124:Conserved Sequence; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007313:Insecta / Q000166:cytology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry*; D014616:Vaccinia virus / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D014443:Tyrosine; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases