Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides (immunologyymajor histocompatibility complexypeptide binding)

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DR1 are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR–peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations. Major histocompatibility complex (MHC) class II molecules present peptide antigens to T cells in the generation of an immune response (1). Antigenic peptides are derived from exogenous proteins, which are processed into peptide fragments by antigen-presenting cells. MHC class II molecules also bind self-peptides derived from cellularly produced proteins. Self-peptides isolated from purified class II molecules, including HLA-DR1 (2, 3), vary in length from '13 to 25 amino acids. This length variability contrasts with the shorter peptides found associated with MHC class I molecules, which are generally 9–11 amino acids long (4–6). For MHC class I molecules the N and C termini of peptides are typically bound in pockets at each end of the peptide-binding site (7–9), whereas for the MHC class II molecules, HLA-DR1 and HLA-DR3, peptides are free to extend out both ends of the binding site. Instead of the network of hydrogen bonds found between the MHC class I molecule and the peptide N and C termini, class II has evolved an alternative hydrogen-bonding interaction along the length of the peptide main chain (10, 11), allowing the termini to extend out of the binding site. This hydrogen-bonding scheme provides interactions with mainchain atoms, which are present in all peptides, and is likely important for the tight binding of peptides of variable sequence (12). Analysis of the electron density corresponding to the mixture of self-peptides bound to MHC class I molecules has provided insights into general aspects of the MHC class I–peptide interaction. In the case of HLA-B27 (8, 9, 13), this provided the first interpretation of the MHC–peptide interactions, whereas for HLA-Aw68 (14), the absence of electron density for the central peptide residues indicated significant conformational heterogeneity, subsequently observed in five different HLA-A2–single peptide structures (9). The electron density corresponding to a mixture of self-peptides bound to HLA-DR1 isolated from the LG-2 B-lymphoblastoid cell line has improved during refinement of the HLA-DR1–Staphylococcus aureus enterotoxin B (SEB) complex. The electron density is continuous and interpretable throughout the peptide-binding site for 13 amino acid residues. Side-chain density is observed for most of the amino acid positions corresponding to residues pointing into pockets of the HLA-DR1 molecule. Three peptide amino acid positions that point out of the peptide-binding site show more complex density, suggesting a mixture of possible amino acid side chains and conformations. After the 13th residue, the electron density bifurcates, suggesting that longer peptides become disordered as they exit the peptide-binding site. The clarity of the peptide main-chain and side-chain density suggests a core binding length of 13 amino acids for peptides that bind to HLA-DR1. The peptide conformation, which has f and c angles close to those of a polyproline type II (ppII) helix, is similar to that observed in the crystal structure of an influenza hemagglutinin peptide bound to the HLA-DR1 molecule (11) and the invariant-chain class II-associated Ii peptides (CLIP) peptide complex with HLA-DR3 (15). Six of the potential hydrogen bonds to the peptide main chain involve conserved asparagine residues of the MHC molecule. These conserved asparagines form 9and 11-membered rings, bidentate hydrogen bonds that in combination with MHC pockets may serve to restrict the possible peptide main-chain conformations. The conserved MHC asparagine residues are poised to interact with the helical repeat of the ppII peptide conformation, placing a common conformational constraint on a repeating tripeptide unit, in which the central amino acid always points out of the peptide-binding The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: MHC, major histocompatibility complex; SEB, Staphylococcus aureus enterotoxin B superantigen; ppII, polyproline type II; HA, hemagglutinin; CLIP, class II-associated Ii peptides. **To whom reprint requests should be addressed.