PEPTIDE BINDING TO MHC CLASS-I IS DETERMINED BY INDIVIDUAL POCKETS INTHE BINDING GROOVE

H-2K(b) and HLA-A2 are MHC4 class I molecules with a similar overall s tructure. Important differences between these two class I molecules re side in the structure of the individual pockets in the antigenic-pepti de-binding groove. H-2K(b), which has a deep C pocket, binds specifica lly peptides with a tyrosine or a phenylalanine at position 5. In cont rast, HLA-A2 has a. shallow C pocket, which cannot accommodate large s ide chains at position 5. Site-directed mutagenesis was used to genera te 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) excep t 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 [V SV (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 'aff inity' to H-2K(b)/HLA-A2(C') than to the wild-type H-2K(b), a VSV anal ogue with the tyrosine in position 5 (Tyr5) substituted with an alanin e was found to bind with a similar 'affinity' to both MHC class I mole cules. Computer-aided modelling of the H-2K(b)/HLA-A2(C') complex indi cates that the VSV (52-59) peptide probably binds to the chimeric MHC molecule with the peptide side chain of anchor residue Tyr5 pointing a way from the groove. These results confirm a role of the individual po ckets in determining peptide-binding affinity and specificity and sugg est that this may be accomplished by changes in side-chain orientation .