Induced Fit of an Epitope Peptide to a Monoclonal Antibody Probed with a Novel Parallel Surface Plasmon Resonance Assay*

Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50–67 of the β-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an α-helical conformation. In the folded, peptide-loaded DR1 structure, the β-chain residues 50–67 contain a kinked α-helical segment spanning Glu-52–Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50–67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (koff). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 β-chain.