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PLoS Pathog. 2016 Dec 14;12(12):e1006079. doi: 10.1371/journal.ppat.1006079. eCollection 2016 Dec.

Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

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Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.
Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, Fife Scotland, United Kingdom.
Structural Biology, IBMB-CSIC, Baldiri Reixach, 13-15, Barcelona, Spain.
Georg August University of Göttingen, Department of Structural Chemistry, Tammannstr. 4, Göttingen, Germany.
European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS, Grenoble, France.
European XFEL GmbH, Notkestraße 85, Hamburg, Germany.
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.
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.
ICREA, Pg. Lluis Companys 23, Barcelona, Spain.


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.

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