Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen.
The Ku antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA. DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear. High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding. In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays. Appropriate folding of the p70 protein was crucial for efficient DNA binding. The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609). Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition. Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding. Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding. The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.
Chou
C H
CH
Division of Rheumatology and Immunology, University of North Carolina School of Medicine, Chapel Hill 27599.
Wang
J
J
Knuth
M W
MW
Reeves
W H
WH
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000145:classification / Q000378:metabolism; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D000939:Epitopes; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen