HLA‐DRB1*0901 is a common allele among Asian populations that has been associated with Type 1 Diabetes. However, its peptide binding motif is only partially characterized. This study further defined the binding motif for DR0901. The motif was deduced from the binding affinities of peptides containing amino acid substitutions at anchor positions and confirmed by structural modeling. In agreement with previous studies, pocket 1 preferred aromatic anchor residues while pocket 4 preferred small aliphatic or polar amino acids. In contrast to previous studies, pocket 9 was not completely permissive, as Pro, Asn, and Arg substitutions were unable to bind. The previously uncharacterized pockets 6 and 7 accommodated a wide range of residues with only subtle influences on overall binding. Modeling studies revealed features within the peptide binding pockets consistent with these findings, including a constrained pocket 4 and a uniquely spacious pocket 9. The resulting motif defined T cell epitopes within 27 of 29 novel antigenic peptides identified by tetramer guided epitope mapping. The observed motif also predicted an epitope within the published GAD65 200–217 peptide and other portions of the protein. This work was supported by NIH contract HHSN266200400028C
Peptide binding to class II MHC protein is commonly viewed as a combination of discrete anchor residue preferences for pockets 1, 4, 6/7, and 9. However, previous studies have suggested cooperative effects during the peptide binding process. Investigation of the DRB1*0901 binding motif demonstrated a clear interaction between peptide binding pockets 6 and 9. In agreement with prior studies, pockets 1 and 4 exhibited clear binding preferences. Previously uncharacterized pockets 6 and 7 accommodated a wide variety of residues. However, although it was previously reported that pocket 9 is completely permissive, several substitutions at this position were unable to bind. Structural modeling revealed a probable interaction between pockets 6 and 9 through beta9Lys. Additional binding studies with doubly substituted peptides confirmed that the amino acid bound within pocket 6 profoundly influences the binding preferences for pocket 9 of DRB1*0901, causing complete permissiveness of pocket 9 when a small polar residue is anchored in pocket 6 but accepting relatively few residues when a basic residue is anchored in pocket 6. The beta9Lys residue is unique to DR9 alleles. However, similar studies with doubly substituted peptides confirmed an analogous interaction effect for DRA1/B1*0301, a beta9Glu allele. Accounting for this interaction resulted in improved epitope prediction. These findings provide a structural explanation for observations that an amino acid in one pocket can influence binding elsewhere in the MHC class II peptide binding groove.
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