posters 5th Asia-Pacific NMR Symposium 2013

Elucidation of nonspecific DNA-binding mechanism by quantitative analysis of chemical shift changes (#153)

Tsuyoshi Konuma 1 , Erisa Harada 1 , Takashi Oda 2 , Mamoru Sato 2 , Kenji Sugase 1
  1. SUNTORY foundation for life sciences, Shimamoto, Mishima, OSAKA, Japan
  2. Yokohama City University, Tsurumi, Kanagawa, Japan

Transcription factors locate effectively to their target DNA. It is generally accepted that a protein first binds nonspecifically to DNA, and then searches for a specific site. However, the details of the dynamic search process are still largely unknown. In this study, we have investigated dynamics of POU homeodomain (POUHD) of Oct3/4 upon DNA binding using NMR. Firstly, we titrated 16-bp DNA into POUHD to characterize the conformational changes and binding kinetics upon DNA binding. Interestingly, almost all peaks in HSQC spectra shifted sigmoidally. They could not be fitted to the simple two-state binding model; therefore, nonspecific bound states should be taken into account to explain the titration data. Here, we constructed a new binding model including the nonspecific bound states using the Bloch-McConnell equation, which describes the time evolution of magnetization including the chemical exchange effect. The fitting of the chemical shift changes to the new model yielded kinetic parameters for POUHD binding to DNA. Subsequently, to obtain structural information on the nonspecific bound states, CLEANEX-PM, which determines exchange rates between amide and water protons, 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 α3-helix 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 α3-helix to its specific target site. This mechanism corresponds to the fly-casting model.