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

Abstract

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 and , derived from the third hypervariable region of the I8 chain of DR1 and DR3, respectively, and the DR1-associated selfpeptide SP3, carrying the fluorophor 7-amino-4-methylcoumarin-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 a, 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 p81-helix that normally lines the binding groove. Thus, the described complexes raise additional possibilities for the molecular basis of auto-or alloreactivity.Major histocompatibility complex (MHC) class II antigens are polymorphic peptide receptors that are loaded with peptides in post-Golgi compartments of an antigen-presenting cell (1). The resulting class II MHC-peptide complexes are of great immunological relevance, since their appearance on the surface of an antigen-presenting cell leads to up-or down-regulation of certain CD4+ T-helper cells that are crucial for self-nonself discrimination (2, 3). Class II molecules bear a single peptide-binding site deduced from sequence homologies and far-reaching functional analogies with crystallographically analyzed human class I antigens (4). Thereafter, both subunits contribute four N-terminal strands of a P-pleated sheet and a laterally overlying a-helical structure forming one-half of the proposed binding groove, respectively (5). The tertiary structure of these domains seems to be highly dependent on the existence of an appropriate peptide, as shown with mouse class II molecules (6): intact MHC-self-peptide complexes migrate as compact conformers (52-56 kDa), whereas heat or acid release of peptides results in slowly migrating floppy conformers (62-67 kDa) in SDS gels. Most recently, McConnell and coworkers (7) have succeeded in showin...

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

“…Indeed, evidence for formation of a two-peptide/MHCII intermediate in the absence of DM has previously been reported [19] , [30] . Measurement of fluorescence resonance energy transfer (FRET) from aromatic residues in the MHCII protein to labeled peptide side chains has been previously utilized to monitor peptide binding, complex dissociation, and the formation of a two-peptide/MHCII complex [19] , [30] , [31] . In general, the intermolecular distance between fluorescent donor and acceptor determines the strength of the FRET signal.…”

Interaction of MHC class II molecules with the invariant chain: role of the invariant chain (81–90) region

“…As a second approach, the IR1 sequence (see Figure 1A) was chemically synthesized. Using an in vitro binding assay based on high‐performance size‐exclusion chromatography (Kropshofer et al ., 1993), this IR1 peptide was found unable to compete binding to purified DR1 molecules of an HA(306–318) peptide labelled with the fluorophor AMCA, even at high concentration. In contrast, unlabelled HA(306–318) peptide competed efficiently (data not shown).…”

HLA-B7 β-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses

“…To couple FITC to the peptides, the method by Kropshofer et al 30 was modified. Peptides attached to the solid support were dissolved in 50 mM borate puffer and were mixed with FITC in a 1:3 molar ratio.…”