Peptide Binding to MHC Class I is Determined by Individual Pockets in the Binding Groove

H-2K^b and HLA-A2 are MHC⁴ class I molecules with a similar overall structure. Important differences between these two class I molecules reside in the structure of the individual pockets in the antigenic-peptide-binding groove. H-2K^b, which has a deep C pocket, binds specifically peptides with a tyrosine or a phenylalanine at position 5. In contrast, HLA-A2 has a shallow C pocket, which cannot accommodate large side chains at position 5. Site-directed mutagenesis was used to generate a chimera between the murine H-2K^b and the human HLA-A2 H-2K^b/HLA-A2(C')]. The structure of this chimera is similar to H-2K^b except for the region around the deep C pocket, where residues at positions 9, 97 and 99 were substituted with those bulkier residues from HLA-A2. Peptide binding between this chimera and H-2K^b-binding peptides VSV (52–59), OVA (257–264), and MCMV pp89 (168–176)], revealed that the deep C pocket of H-2K^b was crucial for high-affinity binding. While a peptide, VSV (52–59), was found to bind with severalfold lower 'affinity' to H-2K^b/HLA-A2(C') than to the wild-type H-2K^b, a VSV analogue with the tyrosine in position 5 (Tyr5) substituted with an alanine was found to bind with a similar 'affinity' to both MHC class I molecules. Computer-aided modelling of the H-2K^b/HLA-A2(C') complex indicates that the VSV (52–59) peptide probably binds to the chimeric MHC molecule with the peptide side chain of anchor residue Tyr5 pointing away from the groove. These results confirm a role of the individual pockets in determining peptide-binding affinity and specificity and suggest that this may be accomplished by changes in side-chain orientation.