Kinetic analysis of peptide binding to the TAP transport complex: Evidencefor structural rearrangements induced by substrate binding

The transporter associated with antigen processing (TAP) plays a key role i n the class I major histocompatibility complex (MHC) mediated immune survei llance. It translocates peptides generated by the proteasome complex into t he endoplasmic reticulum (ER) for loading onto MHC class I molecules. At th e cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step fo r peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we emplo yed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the f luorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP com plex is composed of a fast association step followed by a slow isomerizatio n of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this pepti de-induced structural reorganization may reflect the crosstalk of structura l information between the peptide binding site and both nucleotide-binding domains within the TAP complex. (C) 1999 Academic Press.