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Infection by influenza virus results in the stimulation of cytotoxic T lymphocytes specific for killing virally infected cells. Specificity is provided by clonally distributed, hypervariable T-cell receptors on cytotoxic T lymphocytes which react with peptide fragments that are derived from viral proteins expressed in the cytoplasm and 'presented' on the surface of infected cells, bound to class I histocompatibility glycoproteins. Here we describe the structure of the complex between the human class I histocompatibility glycoprotein HLA-Aw68 and the influenza virus nucleoprotein peptide Np 91-99 as determined by X-ray cryocrystallography. Residues at both ends of the peptide are substantially buried in the peptide binding-site, whereas those in the middle of the peptide, P4 to P8, are predominantly exposed and could be recognized directly by T-cell receptors. The extended conformation of the bound viral peptide is remarkably similar to that of a collection of endogenous peptides with a different sequence motif bound to another human allele, HLA-B27. The structure defines in atomic detail the antigenic surface constructed of major histocompatibility complex and viral peptide atoms that is recognized by T-cell receptors.

Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.

Cytotoxic T lymphocytes kill virally infected cells when they detect antigenic fragments presented by class I major histocompatibility complex (MHC) antigens (HLA in humans). The crystal structures of HLA-A2 and HLA-Aw68 reveal that peptide-antigen forms an integral part of the HLA structure, being retained in a prominent groove even after purification and crystallization. Here we report that the heavy chain and beta 2-microglobulin of HLA-A2, after separation and fractionation in denaturants, reassemble efficiently under renaturing conditions only in the presence of MHC-restricted peptides. A complex of heavy chain, beta 2-microglobulin, and viral peptide in the ratio 1:1:1 is formed in up to 46% yield. Reconstitution is not stimulated by either of two peptides not restricted to HLA-A2. The reconstituted complex of HLA-A2 and the influenza virus (B/Lee/40) nucleoprotein peptide, Np (85-94), crystallizes under conditions previously used to crystallize HLA-A2. Peptide-linked folding and assembly suggests mechanisms for the unusual capacity of HLA to bind many peptides of diverse sequence.

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