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Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 22, 6636-6645 p.Article in journal (Refereed) Published
Abstract [en]

The Kaposi's sarcoma-associated herpesvirus protein SOX (shut off and exonuclease) and its Epstein–Barr virus homolog, BGLF5, are active during the early lytic phase and belong to the alkaline nuclease family. Both proteins have been shown to be bifunctional, being responsible for DNA maturation as well as host shutoff at the mRNA level. We present the crystal structure of SOX determined at 1.85 Å resolution. By modeling DNA binding, we have identified catalytic residues that explain the preferred 5'-exonuclease activity of the alkaline nucleases. The presence of a crevice suitable for binding duplex DNA supports a role for herpes alkaline nucleases in recombination events preceding packaging of viral DNA. Direct interaction with dsDNA is supported by oligonucleotide binding data. Mutations specifically affecting host shutoff map to a surface region of the N-terminal domain, implying an essential role in protein–protein interactions, and link the RNase activity of the enzyme to mRNA degradation pathways.

Place, publisher, year, edition, pages
2009. Vol. 276, no 22, 6636-6645 p.
Identifiers
URN: urn:nbn:se:su:diva-25561DOI: 10.1111/j.1742-4658.2009.07374.xISI: 000271057200021OAI: oai:DiVA.org:su-25561DiVA: diva2:199977
Note
Part of urn:nbn:se:su:diva-8288Available from: 2008-10-30 Created: 2008-10-23 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Expression and structure-function characterisation of herpesviral proteins
Open this publication in new window or tab >>Expression and structure-function characterisation of herpesviral proteins
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult.

The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 76 p.
Keyword
Protein production, Herpesvirus, SOX, E. coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-8288 (URN)978-91-7155-755-1 (ISBN)
Public defence
2008-11-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 09:00 (English)
Opponent
Supervisors
Available from: 2008-10-30 Created: 2008-10-23 Last updated: 2010-01-13Bibliographically approved
2. Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses
Open this publication in new window or tab >>Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Structure biology concerns the study of the molecular structures of biological macromolecules, such as proteins, and how these relate to the function. Protein structures are also of importance in structure-based drug design. In this thesis, the work has been carried out in two different projects. The first project concerns structural studies of proteins from the bacterium Escherichia coli and the second of proteins from five different herpesviruses.

 The E. coli project resulted in the structural characterization of three proteins: CaiB, RibD, and YhaK. CaiB is a type-III CoA transferase involved in the metabolism of carnitine. Its molecular structure revealed a spectacular fold where two monomers were interlaced forming an interlocked dimer. RibD, a bi-functional enzyme, catalyzes two consecutive reactions during riboflavin biosynthesis. In an attempt to characterize the mechanism of action of the N-terminal reductase domain, the structure of RibD was also determined in two binary complexes with the oxidized cofactor, NADP+, and with the substrate analogue ribose-5-phosphate. YhaK is a protein of unknown function normally found in low abundance in the cytosol of E. coli and was previously annotated to be a member of the Pirin family. However, some structural features seem to distinguish YhaK from these other Pirin proteins and we showed that YhaK might be regulated by reactive oxygen species.

 The Herpesvirus project resulted in the structural determination of two proteins, the SOX protein and ORF60 from Kaposi’s sarcoma associated herpesvirus (KSHV). SOX, a bi-functional shutoff and exonuclease protein, is involved in the maturation and packaging of the viral genome into the viral capsid and in the host shutoff of cellular proteins at the mRNA level. The SOX structure was also used for modeling DNA binding. The crystallization and preliminary structural studies of ORF60, the small R2 subunit of the ribonucleotide reductase (RNR) from KSHV is also discussed.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 64 p.
Keyword
CaiB, RibD, YhaK, SOX, ORF60, Herpesvirus, E. coli
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-35674 (URN)978-91-7155-995-1 (ISBN)
Public defence
2010-02-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript.Available from: 2010-01-28 Created: 2010-01-19 Last updated: 2010-01-27Bibliographically approved

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