HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3 10 helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.antigen presentation | antigen processing | major histocompatibility proteins | chaperone | protein folding H LA-DM (DM) facilitates peptide exchange on class II MHC (MHC II) proteins, and is required for efficient peptide loading in vivo (1). MHC II molecules assemble in the endoplasmic reticulum with the class II-associated invariant chain chaperone, which is subsequently cleaved by endosomal proteases leaving a short fragment known as CLIP bound in the MHC peptidebinding groove (2). DM facilitates the exchange of CLIP for peptides generated by digestion of endogenous and exogenous proteins, resulting in a library of peptide antigens bound to MHC II proteins that are transported to the cell surface (3, 4). In vitro experiments have corroborated the roles for DM as a peptideexchange factor (5, 6) and as a molecular chaperone that prevents peptide-free MHC II molecules from becoming inactive and from forming aggregates (7,8).The mechanism by which DM mediates these effects has received much attention (7-17). Peptides are released by DM with different rates (18), with DM susceptibility a major factor in whether or not a particular peptide is recognized by the cellular immune system (19-21). Understanding the mechanism of DMmediated peptide release would promote efforts to predict immunogenicity of known and emerging pathogens. Moreover, the mechanism involves catalysis of a protein conformational change (16), and its elucidation would have implications for our understanding of protein-folding processes. However, despite intensive investigation and crystal structures of both MHC II and DM proteins, the mechanism of DM-facilitated MHC-peptide binding and exchange is not understood. Part of the difficulty in developing an understanding of the interaction of DM and MHC II may be because of the presence of multiple conformers of DM (22), as well as MHC II (10, 23). Previous studies using directed screens (11,14) and tethering approaches (24, 25) have identified residues from both DM and MHC II that are critical for the functional interaction. These residues include a cluster of acidic residues on DM (14) and several residues in the vicinity of the P1 pocket of MHC II (9,11,15). Models for the DM-MHC II complex that pla...

BackgroundMajor histocompatibility complex proteins are believed to undergo significant conformational changes concomitant with peptide binding, but structural characterization of these changes has remained elusive.Methodology/Principal FindingsHere we use molecular dynamics simulations and experimental probes of protein conformation to investigate the peptide-free state of class II MHC proteins. Upon computational removal of the bound peptide from HLA-DR1-peptide complex, the α50-59 region folded into the P1-P4 region of the peptide binding site, adopting the same conformation as a bound peptide. Strikingly, the structure of the hydrophobic P1 pocket is maintained by engagement of the side chain of Phe α54. In addition, conserved hydrogen bonds observed in crystal structures between the peptide backbone and numerous MHC side chains are maintained between the α51-55 region and the rest of the molecule. The model for the peptide-free conformation was evaluated using conformationally-sensitive antibody and superantigen probes predicted to show no change, moderate change, or dramatic changes in their interaction with peptide-free DR1 and peptide-loaded DR1. The binding observed for these probes is in agreement with the movements predicted by the model.Conclusion/SignificanceThis work presents a molecular model for peptide-free class II MHC proteins that can help to interpret the conformational changes known to occur within the protein during peptide binding and release, and can provide insight into possible mechanisms for DM action.

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...

T cells generally recognize peptide antigens bound to MHC proteins through contacts with residues found within or immediately flanking the seven-to nine-residue sequence accommodated in the MHC peptide-binding groove. However, some T cells require peptide residues outside this region for activation, the structural basis for which is unknown. Here, we have investigated a HIV Gagspecific T cell clone that requires an unusually long peptide antigen for activation. The crystal structure of a minimally antigenic 16-mer bound to HLA-DR1 shows that the peptide C-terminal region bends sharply into a hairpin turn as it exits the binding site, orienting peptide residues outside the MHC-binding region in position to interact with a T cell receptor. Peptide truncation and substitution studies show that both the hairpin turn and the extreme C-terminal residues are required for T cell activation. These results demonstrate a previously unrecognized mode of MHC-peptide-T cell receptor interaction.antigen presentation ͉ receptors ͉ antigen ͉ protein conformation C lass II MHC proteins are cell-surface glycoproteins that bind antigens in the form of short peptides and present them for recognition to T cell receptors (TCRs) on the cell surface of CD4 ϩ T cells (1). Naturally processed peptides isolated from class II MHC proteins found in antigen-presenting cells are usually 15-25 residues long (2, 3). The central region of these peptides interacts directly with class II MHC proteins, typically with specific recognition of an approximate nine-residue stretch (4). X-ray crystallography of human and murine class II MHC proteins has revealed that peptides bind to the protein in an extended polyproline type II conformation, with several peptide side chains bound into polymorphic pockets that line the peptide-binding groove (5-12). A hydrogen-bond network between the conserved residues on the class II MHC and the peptide main-chain carbonyl and amide groups, independent of the sequence of the peptide, stabilizes the MHC-peptide complex (13), and enforces the polyproline conformation that directs some of the side chains into the MHC pockets and leaves the others accessible for TCR interactions (14). Generally, pockets accommodate the side chains of peptide residues at the P1, P4, P6, and P9 positions, with smaller pockets or shelves in the binding site accommodating the P3 and P7 residues. Minor variations on this theme have been observed, for example, in some complexes the P9 interactions are weak or absent (5, 10). In the canonical conformation, the side chains of residues at positions P Ϫ1, P2, P5, and P8 are solvent-accessible and point toward the TCR, with portions of other side chains and the peptide main chain also exposed for potential TCR interaction in the central region of the complex.The interaction of TCR with MHC-peptide complexes also is expected to be relatively stereotyped, with complementaritydetermining regions (CDRs) from the V␣ and V␤ domain lying across the peptide-MHC complex, typically with CDR3 loops of both vari...