Structure and Dynamics of Antigenic Peptides in Complex with TAP.

The transporter associated with antigen processing (TAP) selectively translocates antigenic peptides into the endoplasmic reticulum. Loading onto major histocompatibility complex class I molecules and proofreading of these bound epitopes are orchestrated within the macromolecular peptide-loading complex, which assembles on TAP. This heterodimeric ABC-binding cassette (ABC) transport complex is therefore a major component in the adaptive immune response against virally or malignantly transformed cells. Its pivotal role predestines TAP as a target for infectious diseases and malignant disorders. The development of therapies or drugs therefore requires a detailed comprehension of structure and function of this ABC transporter, but our knowledge about various aspects is still insufficient. This review highlights recent achievements on the structure and dynamics of antigenic peptides in complex with TAP. Understanding the binding mode of antigenic peptides in the TAP complex will crucially impact rational design of inhibitors, drug development, or vaccination strategies.

Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy.

The human transporter associated with antigen processing (TAP) is a 150 kDa heterodimeric ABC transport complex that selects peptides for export into the endoplasmic reticulum and subsequent loading onto major histocompatibility complex class I molecules to trigger adaptive immune responses against virally or malignantly transformed cells. To date, no atomic-resolution information on peptide-TAP interactions has been obtained, hampering a mechanistic understanding of the early steps of substrate translocation catalyzed by TAP. Here, we developed a mild method to concentrate an unstable membrane protein complex and combined this effort with dynamic nuclear polarization enhanced magic angle spinning solid-state NMR to study this challenging membrane protein-substrate complex. We were able to determine the atomic-resolution backbone conformation of an antigenic peptide bound to human TAP. Our NMR data also provide unparalleled insights into the nature of the interactions between the side chains of the antigen peptide and TAP. By combining NMR data and molecular modeling, the location of the peptide binding cavity has been identified, revealing a complex scenario of peptide-TAP recognition. Our findings reveal a structural and chemical basis of substrate selection rules, which define the crucial function of this ABC transporter in human immunity and health. This work is the first NMR study of a eukaryotic transporter protein and presents the power of solid-state NMR in this growing field.

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. Here we describe an innovative flow cytometric approach to monitor time-resolved ER compartmentalization of antigenic peptides. This assay allows the analysis of distinct primary human immune cell subsets at reporter peptide concentrations of 1 nM. Thus, this ultrasensitive method for the first time permits quantification of TAP activity under close to physiological conditions in scarce primary cell subsets such as antigen cross-presenting dendritic cells.