TY - JOUR
T1 - Functional heme binding to the intrinsically disordered C-terminal region of bach1, a transcriptional repressor
AU - Segawa, Kei
AU - Watanabe-Matsui, Miki
AU - Matsui, Toshitaka
AU - Igarashi, Kazuhiko
AU - Murayama, Kazutaka
N1 - Funding Information:
We acknowledge the support of the Biomedical Research Core of the Tohoku University Graduate School of Medicine. This work was supported by a JSPS Grant-in-Aid for Scientific Research Number 18H04021 and 15H02506 (K.I.) and Research Fellowships for Young Scientists (grant number 16J40189) and Grant-in-Aid for Scientific Research 16K08573 to M. W.-M. We thank Amy Phillips, Ph.D., from Edanz Group (https://www. edanzediting.com/) for editing a draft of this manuscript.
Funding Information:
The authors declare no conflict of interest. However, K.I. and K.M. received partial financial support from Teijin Pharma
Publisher Copyright:
© 2019 Tohoku University Medical Press.
PY - 2018/3
Y1 - 2018/3
N2 - 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.
AB - 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.
KW - Circular dichroism spectroscopy
KW - Cys-Pro motif
KW - Heme
KW - Intrinsically disordered protein
KW - Protein conformation
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U2 - 10.1620/tjem.247.153
DO - 10.1620/tjem.247.153
M3 - Article
C2 - 30853683
AN - SCOPUS:85062719398
VL - 247
SP - 153
EP - 159
JO - Tohoku Journal of Experimental Medicine
JF - Tohoku Journal of Experimental Medicine
SN - 0040-8727
IS - 3
ER -