Evidence for cobinding of self- and allopeptides to human class II major histocompatibility antigen DR1 by energy transfer.
Purified human class II major histocompatibility antigen HLA-DR1 was subjected to high-performance gel filtration with fluorescence detection to investigate simultaneous binding of two classes of peptides: the N-terminally fluoresceinated allopeptides fluorescein isothiocyanate (FITC)-conjugated DR1 beta-(66-78) and FITC-conjugated DR3 beta-(66-78), derived from the third hypervariable region of the beta chain of DR1 and DR3, respectively, and the DR1-associated self-peptide SP3, carrying the fluorophor 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) at the N terminus. By analyzing the dimer-associated fluorescence signals, we measured an interpeptide energy transfer AMCA-->FITC that proved to be peptide-specific: it did not occur after replacement of the allopeptide by the DR1-restricted peptide IM-(18-29) from influenza matrix protein, whereas it was restored by SP3, due to the high homology of SP3 and allopeptide. Transfer analyses with truncated AMCA-SP3 and AMCA-IM-(18-29) are consistent with Leu-3 being a common anchor residue of both peptides that allows an interaction with the hydrophobic specifity pocket around Ala-37 of the alpha 1 domain. This interaction is mirrored by the intrinsic fluorescence of neighboring Trp-43: we found the protein-peptide transfer Trp(DR1)-->AMCA with AMCA-SP3 but with none of the allopeptides. Since each energy transfer affords close proximity of two fluorophors, the following picture emerges: self- or foreign peptides bind to the DR1 binding cleft by occupation of previously described specificity pockets. Simultaneously, allopeptides of the third hypervariable region or homologous peptides may occupy a cryptic binding site by displacing the beta 1-helix that normally lines the binding groove. Thus, the described complexes raise additional possibilities for the molecular basis of auto- or alloreactivity.
Kropshofer
H
H
Department of Biochemistry, University of Tübingen, Germany.
Max
H
H
Kalbacher
H
H
D000595:Amino Acid Sequence; D004735:Energy Transfer; D000939:Epitopes / Q000378:metabolism*; D005456:Fluorescent Dyes; D015798:HLA-DR1 Antigen / Q000378:metabolism*; D015803:HLA-DR3 Antigen / Q000378:metabolism*; D007519:Isoantigens / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation
D000939:Epitopes; D005456:Fluorescent Dyes; D015798:HLA-DR1 Antigen; D015803:HLA-DR3 Antigen; D007519:Isoantigens; D046911:Macromolecular Substances; D010446:Peptide Fragments