title: The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.
abstract: Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, National Research Center for Environmental Health, Neuherberg, Germany; Center for Integrated Protein Science Munich at Chair Biomolecular NMR Spectroscopy, Department Chemie, Technische Universität München, Garching, Germany.
authors.forename: Anders
authors.initials: A
authors.lastname: Friberg
authors.affiliation: Department of Gene Vectors, Hematologikum, Helmholtz Zentrum München, National Research Center for Environmental Health, München, Germany.
authors.forename: Sybille
authors.initials: S
authors.lastname: Thumann
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, National Research Center for Environmental Health, Neuherberg, Germany; Center for Integrated Protein Science Munich at Chair Biomolecular NMR Spectroscopy, Department Chemie, Technische Universität München, Garching, Germany.
authors.forename: Janosch
authors.initials: J
authors.lastname: Hennig
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, National Research Center for Environmental Health, Neuherberg, Germany; Center for Integrated Protein Science Munich at Chair Biomolecular NMR Spectroscopy, Department Chemie, Technische Universität München, Garching, Germany; Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Peijian
authors.initials: P
authors.lastname: Zou
authors.affiliation: Institute of Molecular Immunology, Hematologikum, Helmholtz Zentrum München, National Research Center for Environmental Health, München, Germany.
authors.forename: Elfriede
authors.initials: E
authors.lastname: Nössner
authors.affiliation: Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Ling
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, National Research Center for Environmental Health, Neuherberg, Germany; Center for Integrated Protein Science Munich at Chair Biomolecular NMR Spectroscopy, Department Chemie, Technische Universität München, Garching, Germany; Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
authors.affiliation: Department of Gene Vectors, Hematologikum, Helmholtz Zentrum München, National Research Center for Environmental Health, München, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Kempkes
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: C402641:EBNA-2 protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D050505:Mutant Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014764:Viral Proteins