Functional non-equivalence of ATP-binding cassette signature motifs in the transporter associated with antigen processing (TAP).
The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.
Chen
Min
M
Institute of Biochemistry, Biozentrum, Goethe-University Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany.
Abele
Rupert
R
Tampé
Robert
R
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; C073119:TAP1 protein, human; D000255:Adenosine Triphosphate