The transporter associated with antigen processing (TAP) represents a focal point in the immune recognition of virally or malignantly transformed cells by translocating proteasomal degradation products into the endoplasmic reticulum-lumen for loading of MHC class I molecules. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex and used it to examine the interface between the transmembrane and nucleotide-binding domains (NBD) by cysteine-scanning and crosslinking approaches. Herein, we demonstrate the functional importance of the newly identified X-loop in the NBD in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in a heterodimeric ABC halftransporter complex by cysteine cross-linking. Strikingly, either substrate binding or translocation can be blocked by cross-linking the X-loop to coupling helix 2 or 1, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops and coupling helices in substrate binding, signaling, and transport. membrane protein structure ͉ traffic ATPase ͉ substrate recognition ͉ conformational change ͉ cysteine cross-linking

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotidebinding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.The transporter associated with antigen processing (TAP) 4 plays a prominent role in the antigen-processing pathway via major histocompatibility complex (MHC) class I molecules (1, 2). A fraction of proteasomal degradation products is translocated into the endoplasmic reticulum by the TAP complex and loaded onto MHC class I molecules. Peptide-loaded MHC complexes can pass the endoplasmic reticulum quality control and traffic to the cell surface, where they display their antigenic cargo to cytotoxic T-lymphocytes, which can thereby efficiently recognize and eliminate infected or malignantly transformed cells. TAP belongs to the superfamily of ATP-binding cassette (ABC) transporters, which translocate a very broad spectrum of substrates across membranes (3, 4).The TAP complex forms a TAP1/TAP2 heterodimer; each subunit contains a transmembrane domain (TMD) followed by a cytosolic nucleotide-binding domain (NBD). The translocation mechanism can be dissected into an ATP-independent peptide binding and an ATP-dependent translocation step (5). TAP1 and TAP2 are required and sufficient for both processes (5, 6). Peptide binding is composed of a fast association step followed by slow structural reorganization of the transport complex (7,8). Previous studies demonstrated that peptide binding to the TMDs triggers ATP hydrolysis by the NBDs (9, 10). TAP preferentially binds peptides with a length of 8 -16 amino acids (5). Using combinatorial peptide libraries, the first three N-terminal and the last C-terminal residues were identified as critical for peptide binding, whereas amino acids between these "anchor" positions do not significantly contribute to the substrate recognition (11). Remarkably, TAP can also bind peptides with large bulky side chains, including cross-linkers, fluorophors, or extended side chains (9, 12, 13). Strikingly, however, sterically restricted peptides that bind but are not transported do not stim...

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