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Conformation of peptides bound to the transporter associated with antigen processing (TAP)

Herget, Meike, Baldauf, Christoph, Schölz, Christian, Parcej, David, Wiesmüller, Karl-Heinz, Tampé, Robert, Abele, Rupert, Bordignon, Enrica
Proceedings of the National Academy of Sciences of the United States of America 2011 v.108 no.4 pp. 1349-1354
ABC transporters, antigens, electron paramagnetic resonance spectroscopy, endoplasmic reticulum, immune response, peptides
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.