Elucidation of the nonspecific DNA-binding mechanism of Oct3/4 using NMR

• Project/Area Number: 25840059
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Suntory Foundation for Life Sciences
• Principal Investigator: TSUYOSHI Konuma 公益財団法人サントリー生命科学財団, その他部局等, 研究員 (10631682)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2014: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2013: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: 非特異的DNA結合 / NMR / 蛋白質の精製 / 溶媒のpH

Outline of Final Research Achievements

In this study, we have investigated dynamics of POU homeodomain (POUHD) of Oct3/4 upon DNA binding using NMR. Firstly, we titrated DNA into POUHD to characterize the conformational changes and binding kinetics upon DNA binding. The quantitative analysis of the chemical shift changes yielded kinetic parameters for POUHD binding to DNA. Subsequently, to obtain structural information on the nonspecific bound states, CLEANEX-PM was applied to POUHD in the presence of excess amounts of POUHD over DNA to increase the population of the nonspecific bound states. As a result, the amide protons of the N-terminal loop are protected from solvent by the bound DNA in the nonspecific binding states, whereas those of the helix region remain to be exchangeable with water protons. Therefore, it is suggested that the N-terminal loop first interacts with nonspecific sites of DNA, and then the stable complex with DNA is formed by locating the helix region to its specific target site.

Research Products

• [Journal Article] Solution structure of the ubiquitin-associated (UBA) domain of human autophagy receptor NBR1 and its interaction with ubiquitin and polyubiquitin (2014)
  • Author(s): Erik, W., Morimoto, D., Sugase, K., Konuma, T., Tochio, H., Shirakawa, M.
  • Journal Title: J. Biol. Chem. Volume: -
  • Peer Reviewed
• [Journal Article] Fast and accurate fitting of relaxation dispersion data using the flexible software package GLOVE (2013)
  • Author(s): Sugase, K., Konuma, T., Lansing, J.C., Wright, P.E.
  • Journal Title: J. Biomol. NMR Volume: 56 Issue: 3 Pages: 275-283
  • DOI: 10.1007/s10858-013-9747-5
  • Peer Reviewed
• [Presentation] Elucidation of the nonspecific DNA-binding mechanism of the POU homeodomain using NMR (2014)
  • Author(s): Tsuyoshi Konuma
  • Organizer: Protein Society Meeting
  • Place of Presentation: San Diego, USA
  • Year and Date: 2014-07-27 – 2014-07-30
• [Presentation] Elucidation of nonspecific DNA-binding mechanism by quantitative analysis of chemical shift changes (2013)
  • Author(s): Konuma, T., Harada, E., Oda, T., Sato, M., Sugase, K.
  • Organizer: 5th Asia-Pacific NMR Symposium in conjunction with ANZMAG
  • Place of Presentation: Brisbane Convention and Exhibition Centre
• [Presentation] Extracting information on protein dynamics from crowded NMR spectra using relaxation dispersion difference
  • Author(s): Konuma, T.
  • Organizer: The 10th Korea-Japan Bilateral Symposium on Biological NMR
  • Place of Presentation: Seoul National University
  • Invited