To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for 58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the -subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the ␤ subunit designed to fill the P1 pocket from within the protein (Gly 86 to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.Class II major histocompatibility complex (MHC) 1 proteins bind peptide antigens and present them to CD4 ϩ T-cells, a process crucial to T-cell selection and initiation of T-cell-mediated immune responses (1, 2). They are cell surface glycoproteins composed of highly polymorphic ␣ and ␤ subunits (ϳ30,000 molecular weight for each subunit), in complex with one bound peptide/molecule. The peptide binding site is found on the substantial extracellular portion of the protein and is formed by both ␣ and ␤ subunits (3); in addition, each subunit has one transmembrane span and a small cytoplasmic portion. Three-dimensional structures have been determined for peptide complexes of extracellular domains of several human (HLA-DR) and murine (I-A and I-E) class II MHC proteins (4 -10). In each of the structures, the peptide is bound in an essentially identical extended conformation, similar to a polyproline II-helix or a twisted ␤ strand, with the conformation apparently maintained by a large number of interactions between the peptide main chain and the MHC protein (10, 11). Side chains of the bound peptides project into allele-specific pockets within the overall peptide-binding site.The peptide binding specificities of class II MHC proteins from human, mouse, and other species have been extensively studied (12). Endogenous peptides isolated from class II MHC molecules expressed by antigen presenting cells generally have lengths ranging from 15 to 20 residues, with longer peptides found occasionally (13-15). Class II MHC proteins generally recognize amino acid side chains embedded within a ϳ9-residue stretch of a bound peptide. Within this binding frame, there are relatively strong side chain preferences at several positions and relatively weaker preferences at others. Sequences outside the binding frame have little or no ...

Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M -1 s -1 ) and dissociation (<10 -5 s -1 ) processes. Various linear and branched pathways have been proposed to account for these data. Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins. Our experiments identified an obligate intermediate in the binding reaction. The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change. The same mechanism is observed for different peptide antigens. In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding. The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed.Proteins encoded by the major histocompatibility complex (MHC) 1 gene locus bind peptide antigens and display them at the cell surface for inspection by the immune system as part of the mechanism by which foreign material in the body is recognized and removed (1). Class II MHC proteins generally are found on specialized immune system cells such as B cells, macrophages, and dendritic cells, but they can be expressed by most cell types in response to inflammation or infection (2). Newly synthesized class II MHC R-and -glycoprotein subunits associate with a chaperonin-like invariant chain protein, which places an extended loop in the class II peptide binding site and directs transport to an endosomal compartment (3). Endosomal proteins cleave the invariant chain, and the bound fragment is exchanged for peptides generated from cell-surface and endocytosed proteins, in a poorly characterized process catalyzed by the peptide-exchange factor HLA-DM (4). MHC-peptide complexes then are transported to the cell surface for inspection by T-cell receptors on CD4 + T lymphocytes. Crystal structures have been determined for several human and murine class II MHC proteins in complex with defined peptides (5-13). In each case, the peptide was bound in a polyproline type II-like conformation, with several side chains projecting into specificity-determining pockets within the overall peptide binding groove, and with many additional contacts between the MHC proteins and the main chain of the bound peptide.Initially, in vitro kinetic measurements of peptide binding to purified class II MHC proteins were interpreted in terms of the simple bimolecular reaction shown in Scheme 1 (14): Physical and chemical characterization of purified class II MHC revealed that they carried complex mixtures of tightly bound endogenous peptides (15-19). The stoichiometry of binding for peptide added to these preparations was quite low. Many of the natural peptide ligands had extremely long half-lives, often on the order of ...

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...