ANALYSIS OF ALLELE-SPECIFIC CONTACT SITES OF NATURAL HLA-DR17 LIGANDS

The sequence motif of peptide ligands naturally associated with DR17 h as indicated conserved residues at the relative positions P1-P4-P6-P8. 9 or 10. Eight naturally processed DR17 ligands were synthesized to st udy the role of conserved residues in DR17 binding. In their majority, they showed an excellent ability to bind to purified DR17 molecules. Binding experiments with variant peptides confirmed aspartate as the D R17-specific contact site at P4. In addition, hydrophobic or aromatic residues at P1 and P9, probably interacting with the NH2- and COOH-ter minal pockets, and lysine or chemically related amino acids at P6 were important for binding. A core peptide of 10 amino acids, bordered by the terminal contact sites, is sufficient, although the ability to bin d is reduced approximately 10-fold compared with the binding capacity of the natural ligand. Ala substitution of flanking stretches at eithe r end completely restores the binding capacity to that of the natural ligand. This suggests that regions flanking the peptide core contribut e to the binding strength nonspecifically, i.e., by forming H-bonds to MHC molecules. Natural DR1 and DR12 ligands like HLA-A2 (103-117) and transferrin receptor (140-156) failed to bind to DR17 molecules. Howe ver, substituting leucine for aspartate at P4 transformed DR1 and DR12 ligands into excellent DR17 binders. This conversion, enabled by a si ngle amino acid substitution, emphasizes the importance of aspartate a s the DR17-specific contact site and suggests that terminal contact re sidues are shared among DR1, DR12, and DR17 ligands. In contrast, addi tional aspartates introduced next to the contact site at P4 impaired t he binding capacity. Regarding this specific role of aspartate we expe ct that DR17-specific ligands will be rarely found among ''promiscuous '' peptides binding to several different DR molecules.