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Modulation of the peptide-binding specificity of a single-chain class II major histocompatibility complex.

We designed and expressed a single-chain class II major histocompatibility complex molecule capable of forming a stable complex with an antigenic peptide. The peptide-binding preference of the single-chain (sc) human leukocyte antigen derived from DRB5(*)0101 (DR51) was determined to be similar to that of the authentic one, which requires a bulky hydrophobic residue at position-1 (P1) as a primary anchor. For modulation of the peptide-binding affinity, we modified binding pocket 1 of sc DR51 by site-directed mutagenesis. The relative binding affinity of the engineered sc DR51 for several P1-substituted peptides was measured by competition assaying with a fluorescence labeled peptide. The sc DR51 molecule showed high affinity to the self-peptide derived from myelin basic protein, 87-98 with Phe as the P1 residue (F90F). While reduction of pocket 1 volume (betaG86V) decreased the affinity of F90F, it rather increased the affinity of the Ala-substituted peptide as to the P1 residue (F90A). Through more extensive engineering in the peptide-binding groove of the sc DR51 molecule, it is expected that we can construct sc DR51 variants with various peptide ligand motifs.