Kinetic Analysis of Peptide Binding to the TAP Transport Complex: Evidence for Structural Rearrangements Induced by Substrate Binding Lars Neumann and Robert Tampe Â* Institut fu È r Physiologische Chemie, Philipps-Universita Èt Marburg, Karl-von-Frisch-Str. 1, 35033 Marburg, Germany The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the protea- some complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for sub- sequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive ¯uorophore. Upon binding to TAP, a drastic ¯uorescence quenching of the ¯uorescent sub- strate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the ¯uorescence label cause ¯uorescence quenching. Taken together, this peptide-induced structural reorganization may re¯ect the crosstalk of structural information between the peptide binding site and both nucleo- tide-binding domains within the TAP complex. # 1999 Academic Press Keywords: ABC transporter; antigen processing; antigen presentation; binding kinetic; ¯uorescence quenching *Corresponding author Introduction Cytotoxic T-lymphocytes recognize and elimin- ate virus-infected or malignant cells. This requires the presentation of fragments derived from viral or tumor-speci®c proteins on the cell surface in associ- ation with major histocompatibility complex (MHC) class I molecules (Pamer & Cresswell, 1998). These peptides are generated in the cytosol mainly by proteasomal degradation (Coux et al., 1996; York & Rock, 1996). Subsequently, the MHC- encoded transporter associated with antigen pro- cessing (TAP) translocates these peptides into the endoplasmic reticulum (ER), where chaperone- assisted assembly and loading of MHC class I mol- ecules occurs (Uebel & Tampe Â, 1999). Kinetically stable MHC-peptide complexes can leave the ER to the cell surface, where they are screened by cyto- toxic T-lymphocytes. TAP belongs to the family of ATP binding cas- sette (ABC) transporters, which are characterized by the highly conserved Walker A/B motifs and the C-loop signature (Higgins, 1992). These trans- port proteins are found in prokaryotes, eukaryotes and archea, where they are responsible for trans- port of a very broad spectrum of substrates. Some important members of the ABC-transporter family are the multidrug resistance P-glycoprotein (MDR), the cystic ®brosis transmembrane conductance reg- ulator (CFTR), the oligopeptide transporter of Sal- monella typhimurium, and the pheromone exporter E-mail address of the corresponding author: tampe@mailer.uni-marburg.de Abbreviations used: ABC, ATP binding cassette; AEBSF, 4-(2-Aminoethyl) benzenesulfonyl ¯uoride; ALD, adrenoleukodystrophy; CFTR, cystic ®brosis transmembrane conductance regulator; cps, counts per second; ER, endoplasmic reticulum; HPLC, high performance liquid chromatography; MCAD, medium- chain acyl dehydrogenase; MDR, multidrug resistance; MHC, major histocompatibility complex; NBD, nucleotide binding domain; PBS, phosphate-buffered saline; DTT, 1,4-dithio-DL-threitol; SDS, sodium dodecylsulfate; TAP, transporter associated with antigen processing. Single letter code is used for peptide sequences. Article No. jmbi.1999.3329 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 1203±1213 0022-2836/99/501203±11 $30.00/0 # 1999 Academic Press