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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

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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.

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Abbreviations

CD:

Circular dichroism

EDTA:

Ethylenediaminetetraacetic acid

HSQC:

Heteronuclear single quantum coherence

NMR:

Nuclear magnetic resonance

PNUTS:

Protein phosphatase 1-binding nuclear targeting protein

PP1:

Protein phosphatase 1

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TCEP:

Tris-2-carboxyethyl-phosphine

TFIIS:

Transcription factor IIS

TFIIS LW-motif:

Structural motif of the transcription factor IIS with conserved leucine and tryptophan residues

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Authors and Affiliations

  1. Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK

    Thomas Zacharchenko, Igor Barsukov, Daniel J. Rigden, Daimark Bennett & Olga Mayans

  2. Department of Biology, Universität Konstanz, Universitätstrasse 10, 78457, Constance, Germany

    Olga Mayans

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  1. Thomas Zacharchenko
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  2. Igor Barsukov
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  3. Daniel J. Rigden
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  4. Daimark Bennett
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  5. Olga Mayans
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Correspondence to Thomas Zacharchenko or Olga Mayans.

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Zacharchenko, T., Barsukov, I., Rigden, D.J. et al. 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. Protein J 35, 340–345 (2016). https://doi.org/10.1007/s10930-016-9677-7

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