title: Conformation of peptides bound to the transporter associated with antigen processing (TAP).
abstract: The ATP-binding cassette transporter associated with antigen processing (TAP) plays a key role in the adaptive immune defense against infected or malignantly transformed cells by translocating proteasomal degradation products into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. The broad substrate spectrum of TAP, rendering peptides from 8 to 40 residues, including even branched or modified molecules, suggests an unforeseen structural flexibility of the substrate-binding pocket. Here we used EPR spectroscopy to reveal conformational details of the bound peptides. Side-chain dynamics and environmental polarity were derived from covalently attached 2,2,5,5-tetramethylpyrrolidine-1-oxyl spin probes, whereas 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled peptides were used to detect backbone properties. Dependent on the spin probe's position, striking differences in affinity, dynamics, and polarity were found. The side-chains' mobility was strongly restricted at the ends of the peptide, whereas the central region was flexible, suggesting a central peptide bulge. In the end, double electron electron resonance allowed the determination of intrapeptide distances in doubly labeled peptides bound to TAP. Simulations based on a rotamer library led to the conclusion that peptides bind to TAP in an extended kinked structure, analogous to those bound to MHC class I proteins.
authors.affiliation: Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Herget
authors.forename: Christoph
authors.initials: C
authors.lastname: Baldauf
authors.forename: Christian
authors.initials: C
authors.lastname: Schölz
authors.forename: David
authors.initials: D
authors.lastname: Parcej
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Wiesmüller
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
authors.forename: Enrica
authors.initials: E
authors.lastname: Bordignon
mesh_terms: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D018411:Spodoptera; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D003497:Cyclic N-Oxides; D019151:Peptide Library; D010455:Peptides; D013113:Spin Labels; C073119:TAP1 protein, human; D014867:Water; C037109:2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylic acid