Journal of the Korean Magnetic Resonance Society (한국자기공명학회논문지)

Korean Magnetic Resonance Society (한국자기공명학회)
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Structural assessment of the tetramerization domain and DNA-binding domain of CP2c

  • Jo, Ku-Sung (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Ryu, Ki-Sung (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Yu, Hee-Wan (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Lee, Seu-Na (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Kim, Ji-Hun (College of Pharmacy, Chungbuk National University) ;
  • Kim, Eun-Hee (Protein Structure Group, Korea Basic Science Institute) ;
  • Wang, Chae-Yeon (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Kim, Chan-Gil (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Kim, Chul Geun (Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University) ;
  • Won, Hyung-Sik (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University)
  • Received : 2018.12.01
  • Accepted : 2018.12.14
  • Published : 2018.12.20
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Abstract

Although the transcription factor CP2c has been recently validated as a promising target for development of novel anticancer therapy, its structure has not been solved yet. In the present study, the purified recombinant protein corresponding to the tetramerization domain of CP2c appeared to be well folded, whereas the Elf-1 domain showed a largely unfolded conformation. Particularly, the Elf-1 domain, which contains the putative DNA-binding region, showed a conformational equilibrium between relatively less-ordered and well-ordered conformers. Interestingly, addition of zinc shifted the equilibrium to the relatively more structured conformer, whereas zinc binding decreased the overall stability of the protein, leading to a promoted precipitation. Likewise, a dodecapeptide that has been suggested to bind to the Elf-1 domain also appeared to shift the conformational equilibrium and to destabilize the protein. These results constitute the first structural characterization of the CP2c domains and newly suggest that zinc ion might be involved in the conformational regulation of the protein.

Keywords

JGGMB2_2018_v22n4_119_f0001.png 이미지

Figure 1. Schematic diagram showing the putative domain organization of CP2c. Constructed regions for structural studies in this study are illustrated by gray bars. TAD, transactivation domain; BD, basic domain; TD, tetramerization domain.

JGGMB2_2018_v22n4_119_f0002.png 이미지

Figure 2. 2D-[1H/15N]-TROSY spectrum of CP2c-TD (0.1 mM). Inset shows its far-UV CD spectrum obtained with 20 μM sample.

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Figure 3. Gel-filtration profiles of the purified Elf1D at various concentrations. Inset shows the elution profiles of separated peak-1 and peak-2 fractions.

JGGMB2_2018_v22n4_119_f0004.png 이미지

Figure 4. Monitoring of zinc binding to Elf1D. (A) Far-UV CD spectra of Elf1D (20 μM) in the absence (black) and presence of 2 equimolar zinc (red). (B) 2D-[1H/15N]-TROSY spectra of Elf1D (150 μM) in the absence (black) and presence of 2 equimoloar zinc (green). (C) Gel-filtration elution profiles of Elf1D (70 μM) in the absence (solid line) and presence of 2 equimolar zinc (dashed line for peak-1 fraction and dotted line for peak-2 fraction).

JGGMB2_2018_v22n4_119_f0005.png 이미지

Figure 5. 2D-[1H/15N]-TROSY spectrum of Elf1D (150 μM) in the absence (black) and presence of a binding peptide (300 μM; blue).

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