Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn 2؉ -binding Ring-Bbox region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn 2؉ -binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn 2؉ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities.Ro52 is one of the main autoantigens in Sjögren syndrome, but autoantibodies to this intracellular protein occur also in systemic lupus erythematosus and rheumatoid arthritis (1). The immunodominant epitopes in Ro52 are predominantly localized in the structurally stable regions (2). The Ro52 coiled-coil contains a putative leucine-zipper between residues 211 and 232. Patient-derived monoclonal antibodies that bind to a peptide representing residues 200 -239 (p200) of the Ro52 protein cause accumulating intracellular calcium levels in neonatal cardiomyocytes and relate to the development of congenital heart block (3-5). Another antigenic epitope has been mapped to the N-terminal Zn 2ϩ -binding region of Ro52 (6) and was also suggested to relate to development of congenital heart block (7). However, the detailed location of the epitope in this 15-kDa region is yet unknown. The epitope is presumably conformation-dependent, because antigenicity is only observed in the reduced state (6), and because cysteines are conserved in the suggested Zn 2ϩ -binding sites. The reason why Ro52 is targeted in autoimmune disease is not known to date. Ro52 belongs to the rapidly growing RING/B-box/coiled-coil (RBCC) 2 family, also denoted as TRIM (trip...