The kinetic basis of peptide exchange catalysis by HLA-DM

Abstract

The mechanism by which the peptide exchange factor HLA-DM catalyzes peptide loading onto structurally homologous class II MHC proteins is an outstanding problem in antigen presentation. The peptide-loading reaction of class II MHC proteins is complex and includes conformational changes in both empty and peptide-bound forms in addition to a bimolecular binding step. By using a fluorescence energy transfer assay to follow the kinetics of peptide binding to the human class II MHC protein HLA-DR1, we find that HLA-DM catalyzes peptide exchange by facilitating a conformational change in the peptide-bound complex, and not by promoting the bimolecular MHC-peptide reaction or the conversion between peptide-receptive and -averse forms of the empty protein. Thus, HLA-DM serves essentially as a protein-folding or conformational catalyst. MHC proteins are heterodimeric cell surface proteins that serve as antigen-presenting elements for the cell-mediated immune system. Class II MHC proteins bind peptide antigens produced by endosomal proteolysis and present them at the cell surface for recognition by CD4 ϩ T cells (1, 2). Newly synthesized class II MHC ␣ and ␤ glycoprotein subunits associate with the invariant chain protein that directs transport to an endosomal compartment (3). Endosomal proteins cleave the invariant chain, leaving a small peptide fragment (known as CLIP) bound in the peptide-binding site. CLIP remains in the binding site until it is exchanged for peptides generated from cell-surface or endocytosed proteins in a process facilitated by the peptide exchange factor HLA-DM (4). Peptide-loaded MHC proteins are transported to the surface for presentation to T cells.HLA-DM is important for efficient endosomal cellular loading of most class II MHC allotypes, and in its absence MHC-CLIP complexes accumulate at the surface (5-8). Crystal structures for HLA-DM and its murine equivalent H2-DM reveal that the overall fold of the molecule is similar to other class II MHC proteins (9, 10), except that the usual MHC-peptide binding groove is largely closed by rearrangements of the flanking helices. DM is not believed to bind peptides but rather to interact with the MHC protein to facilitate peptide binding and release. DM interaction sites on a class II MHC protein have been mapped recently (11). The mechanism by which DM acts to facilitate peptide exchange is an outstanding problem in the field.DM catalyzes both peptide release and binding reactions, and exhibits catalytic turnover such that more than one MHC-bound peptide can be exchanged per DM (12)(13)(14). Thus, DM can be considered an enzyme that catalyzes the peptide-binding reaction. Within this formalism, the kinetic parameters K M Ϸ 10 Ϫ6 M and k cat Ϸ 10 min Ϫ1 have been estimated (15). The presence of catalytic turnover indicates that DM cannot act simply by binding to empty or peptide-loaded class II molecules, which would lead to a stoichiometric but not catalytic process. It has been hypothesized that preferential reaction of DM with rapidly dissociating spe...

Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

“…Closer inspection reveals the presence of multiple distinct species of different behavior and stability (12,26,40): (i) stable DR-peptide complexes from which peptides dissociate at very slow rates (half-life of days to weeks); (ii) a transition state induced by DM from which peptide dissociates rapidly; (iii) a labile empty DR conformer that rapidly binds peptide, but is short-lived; (iv) inactive and aggregated forms; and (v) DM-bound empty DR molecules that are stable and bind peptide rapidly.…”

“…In theory, empty Class II MHC molecules could be generated by dissociating bound peptide, but empty molecules tend to aggregate during the prolonged incubation periods that would be required. An alternative approach that has been pursued consists of the production of recombinant molecules refolded from denatured DR chains in the absence of peptide, but the majority of such molecules (ϳ95%) bind peptides only slowly, apparently because conversion to more active conformer(s) is required (25,26).…”

“…According to the first model, DM interacts only with DR-peptide complexes but not empty DR molecules and catalyzes the interconversion between transient and stable DR-peptide complexes (26). The second model proposes that DM interacts with both DR-peptide complexes and empty DR molecules and that DM acts as a molecular chaperone that rescues empty DR molecules at an acidic pH (27,28).…”

HLA‐DM negatively regulates HLA‐DR4‐restricted collagen pathogenic peptide presentation and T cell recognition

“…The peptides that form stable complexes with class II proteins are displayed at the cell surface for recognition by CD4 + T cells. Additionally, studies suggest that DM molecules regulate epitope selection and T cell responses in the context of HLA class II molecules [20][21][22]. Thus, variations in DM expression within the thymus or in the periphery may regulate class II-restricted presentation of epitopes, potentially influencing the activation of autoreactive T cells.…”

Amino-Terminal Flanking Residues Determine the Conformation of a Peptide–Class II MHC Complex

“…The mechanism underlying these disparate functions is under considerable discussion; one model suggests that DM catalyzes a conformational transition from a peptide-bound state to an open state from which the peptide rapidly dissociates, and subsequently stabilizes this open state (22). The interface of interaction between DM and class II, determined by Mellins and colleagues (23,24) for HLA-DM and HLA-DR1 using random mutagenesis, is contained within a single lateral face of each molecule; significantly, it also includes the amino terminus of the bound peptide, implying that this region of the peptide might be of particular importance for DM function.…”