Interaction between HLA-DM and HLA-DR involves regions that undergo conformational changes at lysosomal pH

Abstract

Antigenic peptide loading of major histocompatibility complex class II molecules is enhanced by lysosomal pH and catalyzed by the HLA-DM molecule. The physical mechanism behind the catalytic activity of DM was investigated by using time-resolved f luorescence anisotropy (TRFA) and f luorescence binding studies with the dye 8-anilino-1-naphthalenesulfonic acid (ANS). We demonstrate that the conformations of both HLA-DM and HLA-DR3, irrespective of the composition of bound peptide, are pH sensitive. Both complexes reversibly expose more nonpolar regions upon protonation. Interaction of DM with DR shields these hydrophobic domains from the aqueous environment, leading to stabilization of the DM and DR conformations. At lysosomal pH, the uncovering of additional hydrophobic patches leads to a more extensive DM-DR association. We propose that DM catalyzes class II peptide loading by stabilizing the low-pH conformation of DR, favoring peptide exchange. The DM-DR association involves a larger hydrophobic surface area with DR͞class II-associated invariant chain peptides (CLIP) than with stable DR͞peptide complexes, explaining the preferred association of DM with the former. The data support a release mechanism of DM from the DM-DR complex through reduction of the interactive surface, upon binding of class II molecules with antigenic peptide or upon neutralization of the DM-DR complex at the cell surface.Presentation of peptides derived from exogenous proteins predominantly occurs in the context of heterodimeric major histocompatibility complex (MHC) class II molecules (1). Binding of these peptides probably takes place in specialized intracellular vesicles, called MHC class II compartments (MIICs; ref. 2). Class II molecules are directed to the MIICs after dimerization in the endoplasmic reticulum and complexing to the invariant chain (Ii) (3). Before binding of internalized peptides, Ii is proteolytically removed, leaving a nested set of peptides (termed CLIP, for class II-associated invariant chain peptides) in the peptide-binding cleft of the class II dimer (4, 5). Subsequent removal of CLIP allows binding of the peptides that are eventually presented to the immune system.The process of peptide loading in the MIICs has not been unravelled, but the lysosomal pH value of the MIICs helps to promote peptide loading of class II molecules (6, 7). For instance, low pH increases protease activity, required both for processing of protein antigen (8) and for Ii proteolysis (9). In addition, CLIP removal from class II is facilitated by low pH (10). Finally, pH affects the mechanism of peptide binding itself (11), possibly through protonation of acidic residues involved in peptide complexing in the class II binding cleft (12) or through a conformational change in the class II dimer (13,14). The HLA-DM molecule is another important component for peptide loading. Cell lines mutated for HLA-DM, or transgenic mice lacking the equivalent murine molecule, accumulate class II͞CLIP complexes at the cell surface (15-18). HLA-DM, ...

Secondary Structure Composition and pH-dependent Conformational Changes of Soluble Recombinant HLA-DM

“…Hydrophobic dye binding of HLA-DM also increases at pH 5, but the effects seem modest compared with those previously seen for classical class II molecules (28,40). 2 We therefore wished to determine the nature and extent of any changes in HLA-DM conformation between neutral and endosomal pH.…”

“…Antibody-blocking experiments and the characterization of a HLA-DR mutant defective for DM binding suggest that a specific face of the class II molecule is involved in DM interactions (16,24,39). Based on hydrophobic dye binding studies, it has been proposed that the interaction involves hydrophobic sites that are buried at the DM-class II interface (40).…”

Evidence for a human leucocyte antigen‐DM‐induced structural change in human leucocyte antigen‐DOβ

“…25 Since the interaction of DM with DR reduces ANS binding to both molecules, it was postulated that the surface of contact is comprised of pH-sensitive regions on both proteins. 7,24,25 In line with this hypothesis and with the critical role of the peptide N-terminal region, Mellins and coworkers recently proposed a model for the DR-DM interaction based on elegant functional mutagenesis data. 27,28 Another non-classical MHC-related molecule, HLA-DO (DO), was shown to accumulate in MIIC-like compartments.…”

“…22,23 For DM, many experiments also suggest that protonation in the endocytic pathway results in minor, reversible structural changes exposing hydrophobic regions of the heterodimer. [24][25][26] Ullrich et al used 8-anilino-1-naphthalenesulphonic acid (ANS), a fluorescent dye binding to hydrophobic protein patches, to demonstrate subtle pH-induced changes in purified DM and DR molecules. 25 Since the interaction of DM with DR reduces ANS binding to both molecules, it was postulated that the surface of contact is comprised of pH-sensitive regions on both proteins.…”

“…[24][25][26] Ullrich et al used 8-anilino-1-naphthalenesulphonic acid (ANS), a fluorescent dye binding to hydrophobic protein patches, to demonstrate subtle pH-induced changes in purified DM and DR molecules. 25 Since the interaction of DM with DR reduces ANS binding to both molecules, it was postulated that the surface of contact is comprised of pH-sensitive regions on both proteins. 7,24,25 In line with this hypothesis and with the critical role of the peptide N-terminal region, Mellins and coworkers recently proposed a model for the DR-DM interaction based on elegant functional mutagenesis data.…”