Peptides induce ATP hydrolysis at both subunits of the transporter associated with antigen processing.
The transporter associated with antigen processing (TAP) plays a key role in the adaptive immune response by pumping antigenic peptides into the endoplasmic reticulum for subsequent loading of major histocompatibility complex class I molecules. TAP is a heterodimer consisting of TAP1 and TAP2. Each subunit is composed of a transmembrane domain and a nucleotide-binding domain, which energizes the peptide transport. To analyze ATP hydrolysis of each subunit we developed a method of trapping 8-azido-nucleotides to TAP in the presence of phosphate transition state analogs followed by photocross-linking, immunoprecipitation, and high resolution SDS-PAGE. Strikingly, trapping of both TAP subunits by beryllium fluoride is peptide-specific. The peptide concentration required for half-maximal trapping is identical for TAP1 and TAP2 and directly correlates with the peptide binding affinity. Only a background level of trapping was observed for low affinity peptides or in the presence of the herpes simplex viral protein ICP47, which specifically blocks peptide binding to TAP. Importantly, the peptide-induced trapped state is reached after ATP hydrolysis and not in a backward reaction of ADP binding and trapping. In the trapped state, TAP can neither bind nor exchange nucleotides, whereas peptide binding is not affected. In summary, these data support the model that peptide binding induces a conformation that triggers ATP hydrolysis in both subunits of the TAP complex within the catalytic cycle.
Chen
Min
M
Institute of Biochemistry, Biocenter, 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; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D017874:Immediate-Early Proteins / Q000502:physiology; D007313:Insecta; D008861:Microsomes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D013696:Temperature; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D003432:Cross-Linking Reagents; C105843:ICP47 protein, Herpes simplex virus; D017874:Immediate-Early Proteins; D010455:Peptides; C073119:TAP1 protein, human; D014764:Viral Proteins; C071692:TAP2 protein, human; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases