Structures and partial functional studies of two Pyrococcus furiosus proteins beyond high throughput structural genomics effort
MetadataShow full item record
Structural genomics initiatives aim to determine the three-dimensional structure of all proteins. The Southeast Collaboratory for Structural Genomics (SECSG) focuses on Pyrococcus. furiosus, a hyperthermophillics archaeon, as a major model organism using a two-tiered approach to achieve this ultimate goal. The SECSG crystallomics group was formed in tier-2 approach to rescue tier-1 failed target. Structural studies of PF0863 and PF0864 from Pyrococcus furiosus were initiated in the context of the structural genomics effort, but failed in the high-throughput structural determination. By using the non-high throughput salvaging practice, both protein structures were solved by single wavelength anomalous dispersion (SAD) method. The structure of PF0863 represents the first released structure of the CYTH domain superfamily defined from protein family (Pfam) database. The biochemical experimental results show that PF0863 has the activity as the nucleotidase. The structural analysis shows high three dimensional similarities between PF0863 and yeast RNA triphasphatase belonged to the mRNA-triPase family. This structural similarity urges us to reconsider the definitions of these two protein families and their evolution relationship. The structure of PF0864 is the second available structure of transcriptional regulator belonged to Lrp/AscC family from P. furiosus. PF0864 can specifically bind to the DNA fragment upstream of the whole operon containing genes PF0865, PF0864, and PF0863, suggesting its autoregulatory function to its own gene expression and members in the same operon under environmental changes. By using genomic context comparison and structure docking methods, PF0863 and PF0864 show a high possibility to form a complex. However, the experimental methods using the size exclusion chromatography followed by native PAGE gel analysis and co-expression followed by GST-pull down assay did not show detectable interactions between PF0863 and PF0864.