| 1. |
- Dahlroth, Sue-Li, et al.
(författare)
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Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:22, s. 6636-6645
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
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| 4. |
- Gurmu, Daniel, et al.
(författare)
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Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi's sarcoma-associated herpesvirus (KSHV)
- 2010
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 66, s. 734-737
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The alpha-herpesviruses and gamma-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the beta-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi's sarcoma-associated gamma-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 angstrom resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 angstrom, alpha = 90, beta = 106.7, gamma = 90 degrees. The data set collected was 95.3% complete, with an R-merge of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%.
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| 5. |
- Gurmu, Daniel, 1974-
(författare)
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Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Gurmu, Daniel, et al.
(författare)
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The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins
- 2009
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 74:1, s. 18-31
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Tidskriftsartikel (refereegranskat)abstract
- YhaK is a protein of unknown function found in low abundance in the cytosol of Escherichia coli. DNA array studies have revealed that YhaK is strongly up-regulated by nitroso-glutathione (GSNO) and also displays a 12-fold increase in expression during biofilm growth of E. coli 83972 and VR50 in human urine. We have determined the YhaK crystal structure and demonstrated that in vitro YhaK is a good marker for monitoring oxidative stresses in E. coli. The YhaK protein structure shows a bicupin fold where the two cupin domains are crosslinked with one intramolecular disulfide bond (Cys10 to Cys204). We found that the third cysteine in YhaK, Cys122, is oxidized to a sulfenic acid. Two chloride ions are found in the structure, one close to the reactive Cys122, and the other on a hydrophobic surface close to a symmetry-related molecule. There are major structural differences at the N-terminus of YhaK compared with similar structures that also display the bicupin fold (YhhW and hPirin). YhaK showed no quercetinase and peroxidase activity. However, reduced YhaK was very sensitive to reactive oxygen species (ROS). The complete, functional E. coli glutaredoxin or thioredoxin systems protected YhaK from oxidation. E. coli thioredoxin reductase and NADPH produced ROS and caused oxidation and oligomerization of reduced YhaK. Taken together, we propose that YhaK is the first of a new sub-class of bicupins that lack the canonical cupin metal-binding residues of pirins and may be involved in chloride binding and/or sensing of oxidative stress in enterobacteria.
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