MHC-I epitope presentation to CD8 + T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptideloading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.MHC-I | tapasin | peptide editing | TAPBPR

MHC class I downregulation represents a significant challenge for successful T cell–based immunotherapy. T cell epitopes associated with impaired peptide processing (TEIPP) constitute a novel category of immunogenic Ags that are selectively presented on transporter associated with Ag processing–deficient cells. The TEIPP neoepitopes are CD8 T cell targets, derived from nonmutated self-proteins that might be exploited to prevent immune escape. In this study, the crystal structure of H-2Db in complex with the first identified TEIPP Ag (MCLRMTAVM) derived from the Trh4 protein has been determined to 2.25 Å resolution. In contrast to prototypic H-2Db peptides, Trh4 takes a noncanonical peptide-binding pattern with extensive sulfur–π interactions that contribute to the overall complex stability. Importantly, the noncanonical methionine at peptide position 5 acts as a main anchor, altering only the conformation of the H-2Db residues Y156 and H155 and thereby forming a unique MHC/peptide conformer that is essential for recognition by TEIPP-specific T cells. Substitution of peptide residues p2C and p5M to the conservative α-aminobutyric acid and norleucine, respectively, significantly reduced complex stability, without altering peptide conformation or T cell recognition. In contrast, substitution of p5M to a conventional asparagine abolished recognition by the H-2Db/Trh4-specific T cell clone LnB5. We anticipate that the H-2Db/Trh4 complex represents the first example, to our knowledge, of a broader repertoire of alternative MHC class I binders.