Crystal Structure of a Non-canonical Low-affinity Peptide Complexed with MHC Class I: A New Approach For Vaccine Design Vasso Apostolopoulos 1,2 *, Minmin Yu 1 , Adam L. Corper 1 , Luc Teyton 3 Geoffrey A. Pietersz 3 , Ian F. C. McKenzie 3 and Ian A. Wilson 1,4 * 1 Department of Molecular Biology, BCC-206 The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037, USA 2 The Austin Research Institute Immunology and Vaccine Laboratory, Studley Road Heidelberg, Vic. 3084 Australia 3 Department of Immunology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037, USA 4 Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide- binding groove. Peptides that do not contain these canonical anchor resi- dues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A ˚ resolution of an immunogenic, low-affinity peptide from the tumor-asso- ciated antigen MUC1, bound to H-2K b . Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This MUC1/K b structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular inter- actions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines. q 2002 Elsevier Science Ltd. All rights reserved Keywords: tumor peptide; non-canonical anchor motif peptides; MUC1; tumor immunotherapy; H-2K b *Corresponding authors Introduction Antigen recognition by T-cells is central to the generation and regulation of an effective immune response. The first step in T-cell generation is the uptake and presentation of antigenic peptides by MHC molecules on antigen-presenting cells. Crystallographic studies of MHC class I molecules have revealed that the amino and carboxy termini of high affinity 8–10-mer peptides (P1–Pn ) are tethered in the groove by conserved hydrogen bond networks. 1–3 The side-chains of bound pep- tides differentially occupy various specificity pockets (A–F) that form in the binding groove between the long a1 and a2 helices and the b-sheet platform. 4 By determining the amino acid sequence of peptides eluted from purified class I molecules, each MHC allele was found to have preferences for particular amino acids at (usually) two particular positions (anchors) in the peptide. 5,6 However, this method fails to identify low-affinity peptides that are lost prior to elution. Anchors for most class I alleles are found at P2 (B pocket) and at the usual C-terminal residue P9 (F pocket). However, H-2K b binds equally well to canonical 8-mer and 9-mer peptides containing preferred anchors Phe/Tyr for the central P5/6 residues (C pocket), Leu at the C terminus (P8/9) (F pocket), and, in some instances, Tyr at P3 (D pocket). Although these anchor residues are usually necessary for high-affinity binding and stabilization of individual MHC isotypes, the presence of appropriate anchors is not a sufficient prerequisite to define production of high-affinity 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail addresses of the corresponding authors: v.apostolopoulos@ari.unimelb.edu.au; wilson@scripps.edu Abbreviations used: CTL, cytotoxic T lymphocyte; M-FP, MUC1 fusion protein containing five VNTR repeats conjugated to oxidized mannan; MPD, 2-methyl- 2,4-pentanediol; VNTR, variable number of tandem repeats. doi: 10.1016/S0022-2836(02)00196-1 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 318, 1293–1305