title: The fellowship of the RING: the RING-B-box linker region interacts with the RING in TRIM21/Ro52, contains a native autoantigenic epitope in Sjögren syndrome, and is an integral and conserved region in TRIM proteins.
abstract: Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn(2+)-binding region, which comprises an N-terminal RING followed by a B-box, separated by a approximately 40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn(2+) increases its stability. Regions stabilized by Zn(2+) binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.
authors.affiliation: Molecular Biotechnology, IFM, Campus Valla, Linköping University, SE-581 83 Linköping, Sweden.
authors.forename: Janosch
authors.initials: J
authors.lastname: Hennig
authors.forename: Anders
authors.initials: A
authors.lastname: Bresell
authors.forename: Martina
authors.initials: M
authors.lastname: Sandberg
authors.forename: Klaus D M
authors.initials: KD
authors.lastname: Hennig
authors.forename: Marie
authors.initials: M
authors.lastname: Wahren-Herlenius
authors.forename: Bengt
authors.initials: B
authors.lastname: Persson
authors.forename: Maria
authors.initials: M
authors.lastname: Sunnerhagen
mesh_terms: D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000145:classification / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000145:classification / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D054829:RING Finger Domains; D012261:Ribonucleoproteins; D012859:Sjogren's Syndrome / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D000099207:SS-A Antigen
chemical_list: D001324:Autoantigens; D004268:DNA-Binding Proteins; D000939:Epitopes; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D015032:Zinc; D000099207:SS-A Antigen