Key residues revealed in a major conformational epitope of the U1-70K protein

Epitopes depending on three-dimensional folding of proteins have during rec ent years been acknowledged to be main targets for many autoantibodies. How ever. a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of im munodominant epitopes of the U1-70K protein, the major autoantigen recogniz ed by human ribonucleoprotein (RNP)-positive sera, we have used diversely m utated recombinant Drosophila melanogaster 70K proteins as antigens in assa ys for human anti-RNP antibodies. Thus. the contribution of individual amin o acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be r econstituted rather than obliterated was enabled. Our results reveal that a mino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119-1 26 also appear to be of utmost importance for recognition. These results ar e discussed in relation to structural models of RNA-binding domains, and te rtiary structure modeling indicates that the residues 119-126 are situated at easily accessible positions in the end of an a-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possib le to analyze other immunodominant regions in which protein conformation ha s a strong impact.