Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets.

Presentation of peptides on major histocompatibility complex class I (MHC I) is essential for the establishment and maintenance of self-tolerance, priming of antigen-specific CD8(+) T cells and the exertion of several T-cell effector functions. Cytosolic proteasomes continuously degrade proteins into peptides, which are actively transported across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). In the ER lumen antigenic peptides are loaded onto MHC I, which is displayed on the cell surface.

In-situ spin labeling of his-tagged proteins: distance measurements under in-cell conditions.

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin-labeled chemical recognition unit for switchable and concomitantly high affinity binding to His-tagged proteins was synthesized. In combination with an orthogonal site-directed spin label, this novel spin probe, Proxyl-trisNTA (P-trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR.

Molecular architecture of the MHC I peptide-loading complex: one tapasin molecule is essential and sufficient for antigen processing.

The loading of antigen-derived peptides onto MHC class I molecules for presentation to cytotoxic T cells is a key process in adaptive immune defense. Loading of MHC I is achieved by a sophisticated machinery, the peptide-loading complex (PLC), which is organized around the transporter associated with antigen processing (TAP) with the help of several auxiliary proteins. As an essential adapter protein recruiting MHC I molecules to TAP, tapasin catalyzes peptide loading of MHC I.

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

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.

Single residue within the antigen translocation complex TAP controls the epitope repertoire by stabilizing a receptive conformation.

The recognition of virus infected or malignantly transformed cells by cytotoxic T lymphocytes critically depends on the transporter associated with antigen processing (TAP), which delivers proteasomal degradation products into the endoplasmic reticulum lumen for subsequent loading of major histocompatibility complex class I molecules. Here we have identified a single cysteinyl residue in the TAP complex that modulates peptide binding and translocation, thereby restricting the epitope repertoire. Cysteine 213 in human TAP2 was found to be part of a newly uncovered substrate-binding site crucial for peptide recognition.