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

Biochemistry. 2006 Jun 20;45(24):7605-16. doi: 10.1021/bi060308o.


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).

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / chemistry*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • ATP-Binding Cassette Transporters / chemistry*
  • ATP-Binding Cassette Transporters / genetics
  • Adenosine Triphosphatases / analysis
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism*
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • Conserved Sequence
  • Dimerization
  • HeLa Cells
  • Humans
  • Hydrolysis
  • Insecta / cytology
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sequence Homology, Nucleic Acid
  • Trypsin / pharmacology
  • Tyrosine / chemistry*
  • Vaccinia virus / metabolism


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • ATP-Binding Cassette Transporters
  • Tyrosine
  • Adenosine Triphosphate
  • Trypsin
  • Adenosine Triphosphatases