Segawa Kei, Watanabe-Matsui Miki, Matsui Toshitaka, Igarashi Kazuhiko, Murayama Kazutaka
Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University.
Pharmaceutical Discovery Research Laboratories, Teijin Pharma Limited.
Tohoku J Exp Med. 2019 Mar;247(3):153-159. doi: 10.1620/tjem.247.153.
Heme is one of the key factors involved in the oxidative stress response of cells. The transcriptional repressor Bach1 plays an important role in this response through its heme-binding activity. Heme inhibits the transcriptional-repressor activity of Bach1, and can occur in two binding modes: 5- and 6-coordinated binding. The Cys-Pro (CP) motif has been determined to be the heme-binding motif of Bach family proteins. The sequence of Bach1 includes six CP motifs, and four CP motifs are functional. With the aim of elucidating the molecular mechanism of heme-Bach1 regulation, we conducted biophysical analyses focusing on the C-terminal region of mouse Bach1 (residues 631-739) which is located after the bZip domain and includes one functional CP motif. UV-Vis spectroscopy indicated that the CP motif binds heme via 5-coordinated bond. A mutant, which included a cysteine to alanine substitution at the CP motif, did not show 5-coordination, suggesting that this binding mode is specific to the CP motif. Surface plasmon resonance revealed that the binding affinity and stoichiometry of heme with the Bach1 C-terminal region were K = 1.37 × 10 M and 2.3, respectively. The circular dichroism spectrum in the near-UV region exhibited peaks for heme binding to the CP motif. No significant spectral shifts were observed in the far-UV region when samples with and without heme were compared. Therefore, disordered-ordered transition such as "coupled folding and binding" is not involved in the Bach1-heme system. Consequently, the heme response of this C-terminal region is accomplished by disorder-disorder conformational alteration.
血红素是细胞氧化应激反应中的关键因素之一。转录抑制因子Bach1通过其血红素结合活性在该反应中发挥重要作用。血红素抑制Bach1的转录抑制活性，且能以两种结合模式发生：五配位结合和六配位结合。已确定半胱氨酸-脯氨酸（CP）基序是Bach家族蛋白的血红素结合基序。Bach1的序列包含六个CP基序，其中四个是有功能的。为了阐明血红素-Bach1调控的分子机制，我们进行了生物物理分析，重点关注小鼠Bach1的C末端区域（631-739位氨基酸残基），该区域位于bZip结构域之后，包含一个有功能的CP基序。紫外-可见光谱表明CP基序通过五配位键结合血红素。一个在CP基序处将半胱氨酸替换为丙氨酸的突变体未显示出五配位，这表明这种结合模式是CP基序特有的。表面等离子体共振显示血红素与Bach1 C末端区域的结合亲和力和化学计量比分别为K = 1.37 × 10 M和2.3。近紫外区域的圆二色光谱显示出血红素与CP基序结合的峰。比较有无血红素的样品时，在远紫外区域未观察到明显的光谱位移。因此，“耦合折叠与结合”等无序到有序的转变不参与Bach1-血红素系统。因此，该C末端区域的血红素反应是通过无序到无序的构象改变来完成的。
