以核磁共振法探討陰道滴蟲Myb2蛋白質和去氧核酸的結合機制

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

蔡丞坤(Cheng-Kun Tsai)

指導教授 ： 黃太煌

國立臺灣師範大學/理學院/物理學系/博士(2009年)

摘要

源於陰道滴蟲的Myb2蛋白質被發現會與特定的MRE2r與MRE2f去氧核酸序列結合,此二序列同時與鐵離子的活性誘導和ap65-1基因轉錄有關.剪裁縮短後的Myb2蛋白質氨基酸長度40-156 (Myb2x)仍保持著與去氧核酸相似的結合能力.利用核磁共振法計算在水溶液中Myb2x蛋白質結構和與MRE2f去氧核酸結合後的複合物結構.結果顯示Myb2x蛋白質在兩者結構中均含三個α螺旋的R2區域和三個α螺旋的R3區域. Myb2x蛋白質裡的第三α螺旋和linker比起與MRE2f去氧核酸結合後的結構,顯得相當鬆散沒有結構性.Myb2x蛋白質與MRE2f去氧核酸結合後的氨基酸骨架變得更受拘束不能自由擺動.結果也顯示兩者MRE2r與MRE2f去氧核酸結合在Myb2x蛋白質上相同的位置,Myb2x蛋白質會與MRE2r或MRE2f去氧核酸分別形成一比一比例結合的複合物.另外,也顯示鐵離子並不會影響Myb2x蛋白質,Myb2xMRE2r蛋白質去氧核酸複合物,和Myb2xMRE2f蛋白質去氧核酸複合物三者的整體結構.也不會影響Myb2x蛋白質與MRE2r或MRE2f去氧核酸結合形成Myb2xMRE2r或Myb2xMRE2f蛋白質去氧核酸兩種複合物. MRE2f去氧核酸與Myb2x蛋白質結合後, ATAC鹼基的化學位移發生明顯變化.根據二維及三維的NOESY實驗及蛋白質側鏈的化學位移擾動變化分析,顯示MRE2f去氧核酸結合至Myb2x蛋白質的位置是分布在蛋白質的N端,R2區域的第三α螺旋,及R3區域的第三α螺旋位置.更甚者,蛋白質的N端是直接接觸去氧核酸的.在蛋白質與去氧核酸之間的NOE訊號由於缺少完整的定義,而至不能獲得精確Myb2x蛋白質與MRE2f去氧核酸結合的複合物結構.然而,卻可以利用B形式的去氧核酸結構,模擬與Myb2x蛋白質的複合物.

關鍵字

核磁共振法 ； Myb2蛋白質 ； 去氧核酸

並列摘要

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.

參考文獻

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