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Conformation of peptides bound to the transporter associated with antigen processing (TAP)
Edited by Harden M. McConnell, Stanford University, Stanford, CA, and approved December 2, 2010 (received for review August 19, 2010)
↵1M.H. and C.B. contributed equally to this work.

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.
- adaptive immune system
- antigenic peptide binding
- peptide conformation
- double electron electron resonance EPR
- site-directed spin labeling
Footnotes
- 2To whom correspondence may be addressed. E-mail: abele{at}em.uni-frankfurt.de or enrica.bordignon{at}phys.chem.ethz.ch.
Author contributions: R.T., R.A., and E.B. designed research; M.H., C.B., C.S., D.P., and E.B. performed research; C.B., D.P., K.-H.W., and R.A. contributed new reagents/analytic tools; M.H., C.B., R.T., R.A., and E.B. analyzed data; and M.H., C.B., R.T., R.A., and E.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012355108/-/DCSupplemental.
Freely available online through the PNAS open access opton.