DNA Recognition Mechanism of Myb2 Protein Derived from Trichomonas vaginalis by NMR technology

• Main Authors: Tsai, Cheng-Kun, 蔡丞坤
• Other Authors: Huang, Tai-huang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/80596090569878157505

博士 === 國立臺灣師範大學 === 物理學系 === 97 === The Myb2 protein from Trichomonas vaginalis was found to interact with specific sequence contexts MRE2r and MRE2f, and is involved in activation of both iron-inducible and growth-related transcription of the ap65-1 gene. The truncated Myb2 protein, Myb2x, spanning amino acid sequence 40-156, has been found to retain similar DNA binding affinity. We have determined Myb2x solution structures on free form and MRE2f-bound form by NMR. Both structures contain three α-helices at R2 domain and three α-helices at R3 domain. In free form the third helix and the linker are more flexible than in complex. Upon binding to MRE2f, the backbone dynamics of Myb2x protein become less flexible. Our results shown that the MRE2r and MRE2f share the same binding site on Myb2x, and the protein to DNA binding ratio is about one to one. On the other hand, the iron does not cause overall structure change in free Myb2x and two complexes, and does not interfere the process of MRE2r/MRE2f binding to Myb2x protein. The larger chemical shift perturbation of MRE2f on Myb2x-bound form appears at four bases ATAC. According to the 2D/3D-filtered NOESY experiments and the perturbation analysis of side-chain chemical shift, the DNA binding site is most likely distributed in the N-terminal, α3-helix of R2 domain, and α6-helix of R3 domain. Moreover, the N-terminal head directly contact to MRE2f DNA. The NOE signal between protein residue and DNA base can’t be well-defined, and the real MRE2f solution structure on Myb2x-bound form isn’t available yet. However, the Myb2x-MRE2f complex model was simulated using solved Myb2x protein structure and modeled B-form MRE2f.