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.
Thomas ZacharchenkoIgor BarsukovDaniel J RigdenDaimark BennettPatricia MurrayPublished in: The protein journal (2017)
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.
Keyphrases
- transcription factor
- dna repair
- magnetic resonance
- endothelial cells
- protein kinase
- amino acid
- dna damage
- cancer therapy
- escherichia coli
- induced pluripotent stem cells
- high resolution
- cell proliferation
- protein protein
- magnetic resonance imaging
- computed tomography
- dna binding
- drug delivery
- oxidative stress
- pluripotent stem cells
- machine learning
- big data
- deep learning
- dna damage response