Kinetic analysis of peptide binding to the TAP transport complex: evidence for structural rearrangements induced by substrate binding.
The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex.
Neumann
L
L
Philipps-Universität Marburg, Karl-von-Frisch-Str. 1, Marburg, 35033, Germany.
Tampé
R
R
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D017951:Antigen Presentation / Q000276:immunology*; D001692:Biological Transport; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D013379:Substrate Specificity; D013816:Thermodynamics
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D000939:Epitopes; D005456:Fluorescent Dyes; D010455:Peptides; D011522:Protons; C071692:TAP2 protein, human; D000255:Adenosine Triphosphate; D019793:Fluorescein