Tohoku J. Exp. Med., 2019, 247, 153-159Heme Binding to Bach1 C-Terminal Region

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Functional Heme Binding to the Intrinsically Disordered
C-Terminal Region of Bach1, a Transcriptional Repressor
Kei Segawa,1,2 Miki Watanabe-Matsui,3,4 Toshitaka Matsui,5 Kazuhiko Igarashi3
and Kazutaka Murayama1,6
Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku
University, Sendai, Miyagi, Japan
2
Pharmaceutical Discovery Research Laboratories, Teijin Pharma Limited, Tokyo, Japan3
Department of Biochemistry, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan
4
Japan Society for the Promotion of Science (JSPS), Tokyo, Japan
5
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, Japan
6
Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research,
Yokohama, Kanagawa, Japan
1

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-coordinatedbinding. 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 aimof 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 notshow 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.
Keywords: circular dichroism spectroscopy; Cys-Pro motif; heme; intrinsically disordered protein; protein
conformation
Tohoku J. Exp. Med., 2019 March, 247 (3), 153-159. © 2019 Tohoku University Medical Press
metabolite bilirubin has antioxidant activity (Chung et al.
2009). These properties suppress apoptosis and inflammatory cytokine production. Thus, inhibition of Bach1 is
expected to induce HO-1 expression to protect cells. Suchan inhibitor represents a potential drug target. Previous
studies on Bach1 knock-out mice have demonstrated theacceleration of biological defense following Bach1 inhibition (Yano et al. 2006; Yamada et al. 2008). Yano et al.
(2006) reported that Bach1 knock-out mice over-expressed
HO-1, and the tissue-protective effect of this enzyme prevented tissue injury in a myocardial ischemia reperfusion
model.
The Bach family proteins Bach1 and Bach2 possess
Broad-Complex, Tramtrack and Bric a brac (BTB) (Ito et

Introduction
The transcriptional repressor Bach1 binds to Maf recognition element (MARE) sequences within the enhancer
and promoter regions of oxidative stress-related genes suchas heme oxygenase (HO)-1, and genes involved in ferritinor heme metabolism (Hintze et al. 2007). Bach1 is inactivated by heme or oxidative stress. When this occurs, transcription of target genes is induced by binding of the Nrf2/
Maf hetero dimer to the MARE sequence which is now
available (Sun et al. 2002; Ishikawa et al. 2005). The HO-1
gene product is the rate-limiting enzyme of heme metabolism. Carbon monoxide that is produced during heme degradation has strong anti-inflammatory actions, and its

Received January 28, 2019; revised and accepted February 19, 2019. Published online March 9, 2019; doi: 10.1620/tjem.247.153.
Correspondence: Kazutaka Murayama, Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical
Engineering, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
e-mail: kazutaka.murayama.d4@tohoku.ac.jp

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al. 2009; Rosbrook et al. 2012) and Basic Leucine Zipper(bZip) domains, separated by long unstructured sequences.
The Bach proteins contain a conserved Cys-Pro (CP) motif,
which has been identified as the heme binding site (Lathrop
and Timko 1993; Zhang and Guarente 1995). There are
two heme binding modes for Bach proteins: 5- and 6-coordination (Hira et al. 2007; Watanabe-Matsui et al. 2011;
Igarashi and Watanabe-Matsui 2014). Of these binding
modes, the axial ligand of 5-coordination is specific to thecysteine in the CP motif. The two modes of heme binding
play significant roles in regulating the intrinsically disordered states of the proteins, as the CP motifs are located in
the unstructured regions. Our previous studies of Bach2
revealed that when heme binding occurs in the intrinsicallydisordered region, the conformation of the region is altered(Watanabe-Matsui et al. 2015; Suenaga et al. 2016).
However, the Bach2 heme-binding region was found to be
an insertion part in comparison between Bach1 and Bach2.
The sequence of Bach1 includes a total of six CP motifs(CP1-CP6). The positions of these CP motifs are conserved
between mouse and human orthologues, although humanBach1 contains an extra CP motif at the Cys140. Of these,
the four CP motifs in the C-terminal side (CP3-CP6) are
functional (Suzuki et al. 2004; Hira et al. 2007). The CP
motif located in the C-terminal moiety of the bZip domain(CP6) is thought to be the site of heme binding (Fig. 1), andthe corresponding region of Bach2 (after the bZip domain)
also includes one CP motif. However, the molecular mechanisms of heme binding and response are still unclear. An
understanding of the heme response of Bach1 is essential toelucidate the molecular mechanisms involved in regulationof the heme-Bach1 system.
Here, we report the biophysical characterization of
Bach1. This study focused on the C-terminal heme-binding
region (631-739), which includes one functional CP motif.
The heme binding and response of Bach1 were analyzed
with spectroscopic measurements. The results suggested

that biophysical response of the Bach1 C-terminal region isa disordered-to-disordered alternation.

Materials and Methods
Preparation of Bach1 samples
The mouse Bach1 DNA fragment encoding residues 631-739(named “Bach1HBR-C”; Bach1 heme-binding region, C-terminal)(Fig. 1) was cloned into a pGEX-6P-1 vector (GE Healthcare;
Buckinghamshire, England). A mutant was prepared, in which
Cys649 was substituted with alanine (named “mCP”). Escherichia
coli BL21 cells (BioDynamics Laboratory Inc., Tokyo, Japan), transformed with the expression construct for the GST-Bach1HBR-C
fusion protein, were grown in Luria Broth medium containing 2%
glucose and 100 µg/mL ampicillin at 37°C until the OD600 reached0.6. After the addition of 1 mM isopropyl β-D-1-thiogalacto­pyrano­side (IPTG), cultures were incubated at 16°C overnight. Cells weresuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2
mM tris[2-carboxyethyl]phosphine [TCEP], 0.1% [w/v] n-dodecyl
β-D-maltopyranoside, and protease inhibitor cocktail), sonicated on
ice and centrifuged at 12,000 × g for 20 min. The supernatant was
incubated with Glutathione Sepharose 4B (GE Healthcare) for 2 h at
4°C with gentle rotation. The resin was then washed with wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 50% glycerol), andincubated with elution buffer (20 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 2 mM TCEP, 20 µL/mL Turbo3C protease (FUJIFILM Wako
Pure Chemical, Osaka, Japan)) with gentle rotation overnight at 4°C.
Supernatant containing the eluted protein was applied to a Mono Q
column (GE Healthcare) that had been pre-equilibrated with 20 mM
Tris-HCl, pH 8.0, 2 mM TCEP. A sodium chloride linear gradient
was used to elute the Mono Q-bound fractions. The fraction containing the protein was concentrated to 2-4 mg/mL using a Vivaspin spincolumn (GE Healthcare) and applied to a Superdex 75 Increase column (GE Healthcare) that had been pre-equilibrated with 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH
7.5, 50 mM NaCl, 2 mM TCEP. Protein solutions were typically concentrated to 1-2 mg/mL.
The 25-amino-acid peptide of Bach1 corresponding to residues
634-658 (Fig. 1) was designed to include the heme-binding CP motif
and synthesized by Greiner Bio-One (Tokyo, Japan). A stock solution

Fig. 1. Diagrammatic representation of the Bach1 constructs and synthesized peptides.
Cysteine-proline motifs are depicted by circle.
BTB, Broad-Complex, Tramtrack and Bric a brac; BZip, Basic Leucine Zipper; mCP, Cys649 to alanine mutated construct; CP, cysteine-proline; WT, wild type.

Heme Binding to Bach1 C-Terminal Region
was prepared by dissolving the peptide in distilled water and adjusting to 1 mg/mL.
Circular dichroism and dynamic light scattering measurements
Protein samples were diluted to 8.6 μM (0.1 mg/mL) in 10 mM
HEPES, pH 7.5, 50 mM NaCl, 2 mM TCEP. Circular dichroism (CD)
spectra were measured with or without 8.6 µM heme (hemin, SigmaAldrich, St. Louis, MO, USA) using a J-805 spectrometer (Jasco,Tokyo, Japan) in the far-UV region (200-250 nm) with a 1 mm pathlength cuvette. Spectra were averaged over four scans.
Measurements were taken from 20 to 60°C with 5°C increment steps.
Measurements in the near-UV region (250-400 nm) were performed
using a 10 mm path-length cuvette. Spectra were measured with 0,
8.6, 17.2, and 25.8 µM hemin at 20°C, and averaged over 16 scans.
Dynamic light scattering (DLS) was performed at 25°C using a
Zetasizer NanoS instrument (Malvern Instruments, Worcestershire,UK). Sample concentrations were adjusted to 0.47 mg/mL and 0.27
mg/mL for Bach1HBR-C wildtype (WT) and mCP, respectively.
Measurements were conducted in triplicate. Data were analyzed with155

the algorithms included in the Zetasizer Nano software.
Surface plasmon resonanceExperiments were performed on a BIAcore™ T200 instrument(GE Healthcare Life Sciences, Pittsburgh, PA, USA) with a Sensor
Chip NTA (GE Healthcare Life Sciences). All analyses were performed at 25°C in HBS-N buffer (GE Healthcare Life Sciences) with
1 mM TCEP. For immobilization of ligand, the Sensor Chip NTA
was activated with 0.5 mM NiCl2 followed by capture of the
C-terminal His6-tagged Bach1HBR-C. As the analyte, 1.25-20 µM
heme solution was injected. An untreated flow cell was used as a reference to correct for bulk response. Experiments were performed in
duplicate. Dissociation constants (KD) were determined by fitting to a
1:1 interaction model, using the Biacore software (GE Healthcare
Life Sciences). Stoichiometry was calculated based on the experimental Rmax value, where stoichiometry = Rmax/(MWA/MWL × RL) (A:analyte, L: ligand, RL: immobilization level of ligand R).

Fig. 2. Structure prediction of Bach1.
(A) The secondary structure elements for mouse Bach1 was predicted by PsiPred. The predicted result is shown for the
C-terminal side of Bach1 (481-739). The predicted secondary structure elements are indicated H: helix, E: strand, and
C: coil, respectively. The prediction confidence values are indicated from 0 (low) to 9 (high). The sequence included inthis study is underlined (631-739). The functional CP motif (CP6) is boxed. (B) Predicted structures of the N-terminal
peptide of Bach1HBR-C (SPEQIQILAKYSASDCPLSFLISEK). Helices are indicated by ribbon representations andthe CP motifs are represented by a ball-and-stick model.

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K. Segawa et al.

Ultraviolet-visible spectroscopic analysis
Spectroscopic analyses were conducted as previously described(Watanabe-Matsui et al. 2011). To estimate heme binding, theUV-Vis spectroscopic measurements were performed at 20°C in 20
mM HEPES, pH 7.0, 50 mM NaCl. The sample cuvette contained 2µM each of Bach1HBR-C (WT or mCP) and bovine serum albumin(BSA), and the reference cuvette contained 40 µM BSA only. Hemin
was added to both the sample and reference cuvettes. In the sample
cuvette, Bach1HBR-C bound heme preferentially, and excess heme
bound to BSA. In the reference cuvette, the heme bound to BSA.
Optical absorption spectra were recorded on a Lambda 45 spectrophotometer (Perkin Elmer, Waltham, US).

Results and Discussion
Secondary structure prediction and amino acid frequencies
Secondary structures can be predicted from the aminoacid sequence with high accuracy (Yang et al. 2018), andthis approach is good initial choice for structure determination of unknown proteins. In the present study, secondary
structure prediction was performed using the PSIPRED
server (Buchan et al. 2013). This approach (Jones 1999)
predicted that the N-terminal region (635-642) of
Bach1HBR-C would form a short helix (Fig. 2A). Analysisof amino acid frequency revealed that Bach1HBR-C contained high amounts of proline (11.0%) and serine (14.7%),
which are enriched in the disordered region (Dunker et al.
2001). In addition, cysteine residues are included with highfrequency (4.6%) in compared with other proteins (Tompa
2002), which likely contribute to the function of Bach1 as aheme-binding protein.
Heme binds to the CP motif
Heme binding was investigated using several experimental methods. Surface plasmon resonance analysis indicated that the dissociation constant (KD) and binding number for Bach1HBR-C WT with heme are 1.37 × 10–5 M and
2.3, respectively. The dissociation constant represents a
relatively low affinity, and other heme-binding proteins
exhibit KD values from 1 × 10–6 M (α1-microglobulin) to 1
× 10–14 M (hemopexin) (Immenschuh et al. 2017). In ourprevious report, we found the KD value for Bach2 withheme to be 1.67 × 10–7 M (Suenaga et al. 2016). The binding number of mCP with heme was not evaluated, as theaffinity was likely to be very low and therefore may not be
detectable.
Heme binding to proteins occurs via 5- and 6-coordination (Hira et al. 2007). We have previously reported that
Bach2 can accept heme in these two binding modes in its
intrinsically disordered region (Watanabe-Matsui et al.
2015). The UV-Vis spectroscopic analysis of the present
study revealed two distinctive peaks at 365 and 428 nm inthe spectrum of Bach1HBR-C WT (Fig. 3). These peaks
relate to 5- and 6-coordinated heme binding, respectively(Hira et al. 2007). However, the mCP spectrum lacked theFig. 3. Ultraviolet-Visible spectroscopic studies.
The difference absorption spectra for wild type (upper, left) and Cys649 mutant (upper, right) are shown. Absorption
shifts at increasing heme concentrations are plotted for 364 nm (lower, left) and 428 nm (lower, right). The open circles
and filled squares are indicated wild type and mutant, respectively.
mCP, Cys649 to alanine mutated construct; WT, wild type.

Heme Binding to Bach1 C-Terminal Region

peak at 365 nm (Fig. 3). The mCP mutant contains a single
cysteine to alanine substitution at Cys649 in the CP motif;
therefore, the CP motif can be concluded to be responsiblefor 5-coordinated heme binding. This finding is supported
by previous reports (Hira et al. 2007). The CP motif-specific heme binding was also observed in the near-UV CD
spectrum. The WT spectrum showed a gently-sloping positive peak around 320 nm (Fig. 4A) and a negative peakaround 380 nm. Both peaks increased with increasing heme
concentration. In contrast, mCP did not show any peaks inthis region (Fig. 4B).
Bach1HBR-C is intrinsically disordered
CD spectrometry is commonly used to assess the secondary structure of proteins (Greenfield 2006). The spectrum in far UV region exhibits specific peaks for the secondary structures, and near-UV region include signals fromsuch as aromatic amino acids, and are sensitive to the overall tertiary structure of the protein (Ranjbar and Gill 2009).
The CD spectra in the far UV region (200-250 nm) are
shown in Fig. 5. Spectra of both WT and mCP appear similar, and the addition of heme did not alter the spectra. In
the present study, the similarities in the spectra of thisregion of WT and mCP samples indicated that the two werein similar conformational states. In addition, althoughheme bound to the two constructs through different binding

157

modes, this did not induce secondary structure changes ineither.
DLS can be used to investigate the hydrodynamic
radius of a protein molecule in solution. The hydrodynamic
radius reflects the conformational state of a protein. The
measured Rh values were 2.91 nm for WT and 2.75 nm for
mCP, respectively. According to Uversky (2002), Rh can be
estimated from the molecular mass for various sample
states, including native (N), molten globule (MG), premolten globule (PMG), urea-unfolded (UU), guanidinium chloride (GdmCl)-unfolded (GU), natively unfolded-coil type
(NU-coil), and natively unfolded-premolten globule type
(NU-PMG). The molecular mass of Bach1HBR-C WT is
11,303.4 Da. The estimated Rh values for each protein state
are shown in Table 1. Comparing these values with themeasured R h values shows good agreement with the
NU-coil value, thus suggesting an intrinsically disordered
protein.
Heme binding does not induce helix formation
To focus on the predicted short helix, we synthesized
the N-terminal peptide of Bach1HBR-C, including one CP
motif (SPEQIQILAKYSASDCPLSFLISEK). De novo
peptide structure prediction was possible for this sequence
using PEP-FOLD 3 (Lamiable et al. 2016), which indicatedten predicted structures. Fig. 2B shows top three structures.

Fig. 4. Circular dichroism spectra in the near-ultraviolet region.
Circular dichroism spectra in the near-ultraviolet region for wild type (A) and mutant (B).
mCP, Cys649 to alanine mutated construct; WT, wild type.

Fig. 5. Circular dichroism spectra in the far-ultraviolet region.
Circular dichroism spectra in the far-ultraviolet region for wild type (left) and mutant (right).
mCP, Cys649 to alanine mutated construct; WT, wild type.

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K. Segawa et al.
Table 1. Hydrodynamic radii estimation for various protein conformational states.

conformational state

N

MG

PMG

UU

GU

NU-coilNU-PMGR h (nm)

1.75

2.00

2.39

2.90

3.01

2.80

2.48

Fig. 6. Far-ultraviolet circular dichroism spectra.
(A) Spectra obtained for the synthesized peptide with/without heme. (B) Temperature shift of spectra obtained for
Bach1HBR-C wild type.
mCP, Cys649 to alanine mutated construct; WT, wild type.

The prediction results suggested that the peptide forms ashort helix at the N-terminal moiety, which is consistent
with the results of secondary structure prediction. However,CD analysis of the peptide showed a decreasing curve withno peaks, similar to the spectrum of Bach1HBR-C (Fig.
6A). Furthermore, comparison of the curves without andwith heme revealed that the addition of heme did not induce
secondary structure, even though the CP motif is located in
the vicinity of the predicted helix (Fig. 2B). When CD
spectra of Bach1HBR-C WT were obtained at different
temperatures (Fig. 6B), only a small shift was observed
even when the temperature was increased up to 60°C.
Definitive validation of the existence of secondary structurein this region is challenging, but the results presented here
imply that there is either no helix or only a partial helix
structure.
Changes in intrinsically disordered states have been
observed as unstructured-structured transitions such“encounter complex” or “coupled folding and folding”(Shammas et al. 2016). These proteins/regions exist in an
unfolded or partially-structured state, transitioning to a
structured form upon binding with their partners. The complex formed by the phosphorylated kinase-inducible domain
and the kinase interacting domain (the pKID-KIX complex)
is one such complex (Sugase et al. 2007). Alone, the pKID
domain adopts an unstructured conformation in solution,
forming α-helices upon binding with KIX. Secondary
structure prediction using PSIPRED or PEP-FOLD suggests the existence of helices, which have been confirmedin the crystal structure (Radhakrishnan et al. 1997). The
predicted structure of Bach1HBR-C suggests similarities, inthat helices are predicted; however, no structure was
detected with biophysical analyses in this case. Although itis the native binding partner of Bach1HBR-C, our results

indicate that heme cannot be the trigger for conformationaltransition. The Bach1-heme system must, therefore, have a
distinctive regulation strategy as an intrinsically disordered
protein. A number of potential binding partners have beensuggested for Bach1 (Li et al. 2018), and so the unstructured-structured transition of Bach1 may be induced byother binding partners.
Conclusions
In this study, the Bach1 C-terminal heme-binding
region was analyzed using biophysical techniques. Thisregion was found to be intrinsically disordered, and able toaccept heme in 5- and 6-coordination binding modes. The
CP motif is responsible for 5-coordinated heme binding, as
has been shown in Bach2. The conformational state of
Bach1HBR-C did not change upon the addition of heme,
and no biological response in terms of unstructured-structured transition was observed. We therefore conclude that
Bach1HBR-C does not undergo unstructured-structured
transition with heme; instead, the heme response of theregion occurs through a disorder-disorder conformational
alteration.

Acknowledgments
We acknowledge the support of the Biomedical ResearchCore 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.

Author Contributions
K.S., M.W-M. and K.M. performed experimental studies.

Heme Binding to Bach1 C-Terminal Region
T.M. and K.I. supervised the experiments. K.S. and K.M. wrote
the manuscript.

Conflict of Interest
The authors declare no conflict of interest. However, K.I.
and K.M. received partial financial support from Teijin PharmaCo., Ltd. through a collaborative research contract.

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