title: Biophysical Analysis of the N-Terminal Domain from the Human Protein Phosphatase 1 Nuclear Targeting Subunit PNUTS Suggests an Extended Transcription Factor TFIIS-Like Fold.
abstract: Human protein phosphatase 1 nuclear targeting subunit (PNUTS) plays critical roles in DNA repair, cell growth and survival. The N-terminal domain of PNUTS mediates interactions with Tox4 and the phosphatase and tensin homolog PTEN, which are essential for the roles of this protein. To study this N-terminal domain, we have established its recombinant overproduction in E. coli utilizing NusA fusion. Upon removal of the tag, the remaining PNUTS sample is soluble and highly pure. We have characterized the domain using circular dichroism and nuclear magnetic resonance and analyzed its sequence using bioinformatics. All data agree in suggesting that the PNUTS N-terminal segment adopts a compact, globular fold rich in α-helical content, where the folded fraction is substantially larger than the previously annotated fold. We conclude that this domain adopts a single fold, likely being an extended form of the transcription factor S-II leucine/tryptophan conserved-motif. Thermal denaturation yielded a melting temperature of ~49.5 °C, confirming the stability of the fold. These findings pave the way for the molecular characterization of functional interactions mediated by the N-terminal region of PNUTS.
authors: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK. fbstz@leeds.ac.uk.
authors: Thomas
authors: T
authors: Zacharchenko
authors: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK.
authors: Igor
authors: I
authors: Barsukov
authors: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK.
authors: Daniel J
authors: DJ
authors: Rigden
authors: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK.
authors: Daimark
authors: D
authors: Bennett
authors: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK. olga.mayans@uni-konstanz.de.|Department of Biology, Universität Konstanz, Universitätstrasse 10, 78457, Constance, Germany. olga.mayans@uni-konstanz.de.
authors: Olga
authors: http://orcid.org/0000-0001-6876-8532
authors: O
authors: Mayans
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D000072417:Protein Domains; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D040681:Structural Homology, Protein; D035602:Transcriptional Elongation Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009687:Nuclear Proteins; C110225:PPP1R10 protein, human; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; C018648:transcription factor S-II
keywords: Circular dichroism; Nuclear magnetic resonance; Recombinant protein overexpression; Secondary structure prediction; Thermal denaturation