Modulation of peptide binding by HLA‐B27 polymorphism in pockets A and B, and peptide specificity of B2703

The results in this study address three aspects of peptide binding to the disease‐associated antigen HLA‐B27 and its modulation by polymorphism: the contribution of major anchor residues 2 and 9, the role of pocket B polymorphism in modulating peptide specificity, and the binding properties of B*2703, a subtype not found to be associated with spondyloarthropathy. Synthetic analogs of peptides naturally presented by B*2705 were used to demonstrate that residue 2 is essential, since Ala2 analogs bound marginally to B*2705, but the specificity of B*2705 for Arg2 is not absolute, and show that the contribution of basic residue 9 to binding was significant, but less than Arg2. The effect of single mutations in the B pocket was to decrease or ‐ with the Glu > Met‐45 mutation ‐ totally shift pocket B specificity for Arg2 towards other residues at this position. This was shown by quantitating the relative binding of Gln2 and Ala2 analogs, and by pool‐sequencing of the peptides bound in vivo to these mutants. Peptides naturally presented by B*2705 apparently bound with a lower affinity to pocket A variants with altered hydrogen bonding to the peptide N terminus, including B*2703. Binding of peptide analogs with changes at positions 2 or 9 suggested that in B*2703 pocket A, interactions are weaker and pocket B interactions are stronger than in B*2705. This can be explained by the effect of the unique His59 change in B*2703 in both pockets. Thus, B*2703 is probably the HLA‐B27 subtype with the most stringent specificity for the Arg2 peptide motif.