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The crystal structure of D212 from sulfolobus spindle-shaped virus ragged hills reveals a new member of the PD-(D/E)XK nuclease superfamily.

ABSTRACT: Structural studies have made significant contributions to our understanding of Sulfolobus spindle-shaped viruses (Fuselloviridae), an important model system for archaeal viruses. Continuing these efforts, we report the structure of D212 from Sulfolobus spindle-shaped virus Ragged Hills. The overall fold and conservation of active site residues place D212 in the PD-(D/E)XK nuclease superfamily. The greatest structural similarity is found to the archaeal Holliday junction cleavage enzymes, strongly suggesting a role in DNA replication, repair, or recombination. Other roles associated with nuclease activity are also considered.

SUBMITTER: Menon SK

PROVIDER: S-EPMC2876643 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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The crystal structure of D212 from sulfolobus spindle-shaped virus ragged hills reveals a new member of the PD-(D/E)XK nuclease superfamily.

Menon Smita K SK Eilers Brian J BJ Young Mark J MJ Lawrence C Martin CM

Journal of virology 20100407 12

Structural studies have made significant contributions to our understanding of Sulfolobus spindle-shaped viruses (Fuselloviridae), an important model system for archaeal viruses. Continuing these efforts, we report the structure of D212 from Sulfolobus spindle-shaped virus Ragged Hills. The overall fold and conservation of active site residues place D212 in the PD-(D/E)XK nuclease superfamily. The greatest structural similarity is found to the archaeal Holliday junction cleavage enzymes, strongl ...[more]

PMID: 20375162

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Project description:BackgroundThe uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity.MethodRecombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study.ResultsWe revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity.ConclusionThis is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.

Dataset Information

The crystal structure of D212 from sulfolobus spindle-shaped virus ragged hills reveals a new member of the PD-(D/E)XK nuclease superfamily.

Publications

The crystal structure of D212 from sulfolobus spindle-shaped virus ragged hills reveals a new member of the PD-(D/E)XK nuclease superfamily.

Similar Datasets

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