Evidence for cobinding of self- and allopeptides to human class II major histocompatibility antigen DR1 by energy transfer.

AUTOR(ES)

Kropshofer, H

RESUMO

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.

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