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 processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.
Uebel
S
S
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Meyer
T H
TH
Kraas
W
W
Kienle
S
S
Jung
G
G
Wiesmüller
K H
KH
Tampé
R
R
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017951:Antigen Presentation; D001665:Binding Sites; D001692:Biological Transport; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D008285:Major Histocompatibility Complex / Q000502:physiology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins; C073119:TAP1 protein, human; C071692:TAP2 protein, human