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

Abstract

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 ␤-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 ␣-helical conformation. In the folded, peptide-loaded DR1 structure, the ␤-chain residues 50 -67 contain a kinked ␣-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 (k off ). 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 ␤-chain.Research on factors influencing molecular recognition is an area of vibrant activity. The molecular basis of specificity for antigen-antibody recognition can involve numerous factors and is not limited to the primary amino acid sequence of the antigenic epitope. The epitope can have a complex structural component in the context of the entire tertiary structure of the antigen. In such a case, an analogous linear variant of the original antigen, for instance, a short peptide, would not be expected to retain much structural information, particularly in the absence of any disulfide bridges. However, antibodies that can recognize short peptides that appear to adopt a defined conformation have previously been described (3, 4).MEM-265 is a mouse monoclonal antibody that recognizes the empty conformation of the human class II MHC protein HLA-DR1. Although the antibody was raised against the denatured ␤ subunit, it appears to recognize a conformational epitope present in the native ␣-␤ heterodimer, which becomes unavailable upon peptide binding (1). Preliminary characterization has mapped the epitope to residues 50 -67 of the ␤ subunit, with residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 being essential for binding. To assess the individual contributions of nonessential residues to binding, we used SpotMatrix SPR 1 to obtain a co...

Characterization of the Functional Epitope on the Urokinase Receptor

“…In the following data evaluation, we therefore initially focused on k off values, which seemed reasonable, as this rate constant furthermore yielded the largest contribution to the global change in the Gibbs free energy of binding (⌬⌬G). The impact of specific side chain deletion primarily on k off rather than on k on is concordant with reports for other protein/protein interactions probed by alanine scanning mutagenesis (46,47). The quality of the data evaluation for these interactions is illustrated in supplemental Fig.…”

Structure-Function Analysis of the Epitope for 4E10, a Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody

“…(Tables 1 and 2). The on rates of the three peptides are very similar, as is typically observed for the structurally similar peptide analogs (1). In contrast, the off rates vary by an order of magnitude, consistent with the various stabilities of the bound peptides as reflected by a well-positioned thioether tether (104-2) versus a poorly positioned thioether tether (74-2) compared to the linear peptide 84-1.…”

Surface plasmon resonance sensors in cell biology: basics and application

“…Here, incident light hits the metal sensor layer from the top passing through the analyte containing low refractive index medium. This eliminates on the one hand the need for a high refractive index prism for every measurement channel and so makes this architecture attractive for high-throughput screening purposes [25]. But on the other hand, it also exhibits the big disadvantage that the exciting light has to incite through the analyte matrix, which will very often interfere with the light in terms of absorption or diffraction rendering a SPR measurement difficult if not impossible.…”

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