Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

Abstract

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4؉ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptideinduced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the ␤ subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change. Major histocompatibility complex (MHC)1 molecules are heterodimeric cell-surface proteins that play an important role in the initiation of antigen-specific immune responses. Class II MHC proteins bind peptides derived from extracellular, endosomal, and internalized cell-surface antigens, and present them at the cell surface for inspection by CD4 ϩ T cells (1). Three-dimensional structures have been determined for peptide complexes of several polymorphic variants of both human and murine class II MHC molecules (reviewed in Ref.2). Both the MHC ␣ and ␤ chains contribute to the peptide binding site, which is made up of a ␤ sheet floor topped by two roughly parallel ␣ helical regions. Each subunit contributes an immunoglobulin-like domain below the peptide binding site, as well as short transmembrane and cytoplasmic domains. Peptides bind in an extended conformation in the groove between the two helices, with ϳ10 residues able to interact with the MHC protein, and the peptide termini extending from the binding site. This conformation, similar to a polyproline type II helix, has a 2.7-residue repeat and appears to be dictated by a network of conserved hydrogen bonding interactions between the MHC and bound peptide (3). The conformation places 4 -6 of the peptide side chains into pockets within the overall groove. The residues lining these pockets vary between allelic variants, providing different peptide-sequence binding specificity. Overall the interaction buries ϳ70% of the peptide surface area in the central region of a bound peptide, leaving the remainder available for interaction with antigen receptors on T cells (4).Although the canonical structure visualized by x-ray crystallography is relatively stereotyped, a number of studies h...

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

“…The MEM-265 hybridoma was originally prepared after immunization of mice with purified, insoluble DR1 ␤-chain (DRB1*0101) expressed in Escherichia coli inclusion bodies and screening of hybridomas for the ability to distinguish empty DR1 from hemagglutinin peptide loaded DR1 using enzyme-linked immunosorbent assay. MEM-265 is one of four similar antibodies thus identified (1).…”

“…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.…”

“…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 comparative kinetic analysis for alanine mutants spanning residues 50 -67 of the epitope.…”

“…Kinetic Analysis-A two-compartment model (11) is used to describe the interaction between an analyte (A) and an immobilized ligand (B) in Equation 1.…”

Conformational Variation of Surface Class II MHC Proteins during Myeloid Dendritic Cell Differentiation Accompanies Structural Changes in Lysosomal MIIC

“…For the statistical analysis the randomness test was used (25,26). The following Ab were used: polyclonal anti HLA-DR rabbit sera raised against purified HLA-DR1(42), MEM-265 mouse IgG2b specific for empty/open HLA-DR molecules (27), rabbit serum against the C-terminal of Ii (28), and PIN1 mAb to the N-terminal of Ii.…”