REQUIREMENTS FOR PEPTIDE BINDING TO THE HUMAN TRANSPORTER ASSOCIATED WITH ANTIGEN-PROCESSING REVEALED BY PEPTIDE SCANS AND COMPLEX PEPTIDE LIBRARIES

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen pro cessing (TAP), where they are subsequently needed for the correct asse mbly of major histocompatability complex molecules. The transport func tion was reconstituted in insect cells by expression of both TAP genes . On the basis of this overexpression system, substrate selection was analyzed in detail by a direct bimolecular peptide binding assay. Comp etition assays with peptide variants, including substitutions of resid ues with alanine or structurally related amino acids, underline the br oad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides a nd scans with bulky, hydrophobic amino acids. Complex nonapeptide libr aries were used to determine the contribution of each residue to stabi lize peptide-TAP complexes. For the first time, this ap proach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transport er.