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Functional Heme Binding to the Intrinsically Disordered C-Terminal Region of Bach1, a Transcriptional Repressor

  • Segawa Kei
    Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University Pharmaceutical Discovery Research Laboratories, Teijin Pharma Limited
  • Watanabe-Matsui Miki
    Department of Biochemistry, Graduate School of Medicine, Tohoku University Japan Society for the Promotion of Science (JSPS)
  • Matsui Toshitaka
    Institute of Multidisciplinary Research for Advanced Materials, Tohoku University
  • Igarashi Kazuhiko
    Department of Biochemistry, Graduate School of Medicine, Tohoku University
  • Murayama Kazutaka
    Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research

Abstract

<p>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 KD = 1.37 × 10–5 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.</p>

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