TY - JOUR
T1 - Biophysical characterization of heme binding to the intrinsically disordered region of Bach1
AU - Segawa, Kei
AU - Watanabe-Matsui, Miki
AU - Tsuda, Kengo
AU - Matsui, Toshitaka
AU - Shirouzu, Mikako
AU - Igarashi, Kazuhiko
AU - Murayama, Kazutaka
N1 - Funding Information:
KI and KM received partial financial support from Teijin Pharma Co., Ltd. through a collaborative research contract.
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 (KI) and Research Fellowships for Young Scientists (Grant No. 16J40189) to M. W.-M. We thank Natasha Beeton-Kempen, Ph.D., from Edanz Group ( www.edanzediting.com/ac ) for editing a draft of this manuscript.
Publisher Copyright:
© 2019, European Biophysical Societies' Association.
PY - 2019/5/1
Y1 - 2019/5/1
N2 - Transcriptional repressor Bach1 plays an important role in antioxidant response. Bach1 function is regulated by heme binding to the four cysteine-proline (CP) motifs in Bach1, which leads to inhibition of its activity. Three of these CP motifs are located N-terminal to the bZip (basic leucine zipper) domain that is responsible for DNA binding. Based on sequence analysis, the region surrounding these CP motifs was expected to be intrinsically disordered. Bach1 is one of few known intrinsically disordered proteins that accept multiple heme molecules for functional regulation, but the molecular mechanisms of heme binding and functional regulation remain unclear. Uncovering these mechanisms is important for understanding Bach1-mediated antioxidant response. Biophysical characterization revealed that 5-coordinated heme binding was unique to the CP motifs within the heme-binding region of Bach1, whereas 6-coordinated binding occurred nonspecifically. Comparison of the wild-type protein and a CP motif mutant indicated that the level of 6-coordinated heme binding was reduced in the absence of 5-coordinated heme binding. Analytical ultracentrifugation showed that the CP motif mutant protein had a more elongated conformation than the wild-type protein, suggesting that cysteines within the CP motifs contribute to intramolecular interactions in Bach1. Thus, heme binding at the CP motifs induces a global conformational change in the Bach1 heme-binding region, and this conformational change, in turn, regulates the biological activity of Bach1.
AB - Transcriptional repressor Bach1 plays an important role in antioxidant response. Bach1 function is regulated by heme binding to the four cysteine-proline (CP) motifs in Bach1, which leads to inhibition of its activity. Three of these CP motifs are located N-terminal to the bZip (basic leucine zipper) domain that is responsible for DNA binding. Based on sequence analysis, the region surrounding these CP motifs was expected to be intrinsically disordered. Bach1 is one of few known intrinsically disordered proteins that accept multiple heme molecules for functional regulation, but the molecular mechanisms of heme binding and functional regulation remain unclear. Uncovering these mechanisms is important for understanding Bach1-mediated antioxidant response. Biophysical characterization revealed that 5-coordinated heme binding was unique to the CP motifs within the heme-binding region of Bach1, whereas 6-coordinated binding occurred nonspecifically. Comparison of the wild-type protein and a CP motif mutant indicated that the level of 6-coordinated heme binding was reduced in the absence of 5-coordinated heme binding. Analytical ultracentrifugation showed that the CP motif mutant protein had a more elongated conformation than the wild-type protein, suggesting that cysteines within the CP motifs contribute to intramolecular interactions in Bach1. Thus, heme binding at the CP motifs induces a global conformational change in the Bach1 heme-binding region, and this conformational change, in turn, regulates the biological activity of Bach1.
KW - CP motif
KW - Cysteine reactivity
KW - Heme
KW - Intrinsically disordered proteins
KW - Protein conformation
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U2 - 10.1007/s00249-019-01364-5
DO - 10.1007/s00249-019-01364-5
M3 - Article
C2 - 30937482
AN - SCOPUS:85065810321
VL - 48
SP - 361
EP - 369
JO - European Biophysics Journal
JF - European Biophysics Journal
SN - 0175-7571
IS - 4
ER -