title: Modulation of the peptide-binding specificity of a single-chain class II major histocompatibility complex.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Kusung-dong, Yusung-gu, Taejon 305-701, Korea. smbyun@mail.kaist.ac.kr
authors.forename: S T
authors.initials: ST
authors.lastname: Kim
authors.forename: S M
authors.initials: SM
authors.lastname: Byun
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D002850:Chromatography, Gel; D017931:DNA Primers; D005822:Genetic Vectors; D006684:HLA-DR Antigens / Q000235:genetics / Q000378:metabolism*; D059847:HLA-DRB5 Chains; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding
chemical_list: D017931:DNA Primers; D006684:HLA-DR Antigens; D059847:HLA-DRB5 Chains; D008024:Ligands; D009842:Oligopeptides; D010446:Peptide Fragments