title: Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.
abstract: Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.
authors.affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.
authors.forename: Sofiya
authors.initials: S
authors.lastname: Fedosyuk
authors.affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.
authors.forename: Gustavo Arruda
authors.initials: GA
authors.lastname: Bezerra
authors.affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Radakovics
authors.affiliation: Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, Fife Scotland, United Kingdom.
authors.forename: Terry K
authors.initials: TK
authors.lastname: Smith
authors.affiliation: Structural Biology, IBMB-CSIC, Baldiri Reixach, 13-15, Barcelona, Spain.|Georg August University of Göttingen, Department of Structural Chemistry, Tammannstr. 4, Göttingen, Germany.
authors.forename: Massimo
authors.identifier: http://orcid.org/0000-0002-8346-9247
authors.initials: M
authors.lastname: Sammito
authors.affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.
authors.forename: Nina
authors.initials: N
authors.lastname: Bobik
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS, Grenoble, France.|European XFEL GmbH, Notkestraße 85, Hamburg, Germany.
authors.forename: Adam
authors.initials: A
authors.lastname: Round
authors.affiliation: San Diego Supercomputer Center, University of California, San Diego, La Jolla, California, United States of America.|Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Lynn F
authors.identifier: http://orcid.org/0000-0002-4562-9027
authors.initials: LF
authors.lastname: Ten Eyck
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dept. of Structural and Computational Biology, Campus Vienna Biocenter 5, Vienna, Austria.|Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, Ljubljana, Slovenia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinović-Carugo
authors.affiliation: Structural Biology, IBMB-CSIC, Baldiri Reixach, 13-15, Barcelona, Spain.|ICREA, Pg. Lluis Companys 23, Barcelona, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Usón
authors.affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.
authors.forename: Tim
authors.identifier: http://orcid.org/0000-0001-8865-5500
authors.initials: T
authors.lastname: Skern
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D000072757:Protein Conformation, beta-Strand; D020836:Protein Structure, Quaternary; D053838:Scattering, Small Angle; D021241:Spectrometry, Mass, Electrospray Ionization; D014616:Vaccinia virus / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C413651:A46R protein, vaccinia virus; D014764:Viral Proteins