Biochemical and Biophysical Research Communications xxx (2017) 1e6

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Biochemical and Biophysical Research Communications

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Structural insights into the dimer-tetramer transition of FabI from

Bacillus anthracis
Hyun Tae Kim a, b, Sulhee Kim b, Byeong Kwan Na a, Jiwoung Chung a, Eunha Hwang c,
Kwang Yeon Hwang b, *
a
Crystalgenomics, Inc., 5F, Tower A, Korea Bio Park 700, Daewangpangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13524, South Korea
b
Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, South Korea
c
Division of Bioconvergence Analysis, Korea Basic Science Institute, 162 Yeongudangiro Ochang, Cheongwongu, Cheongju, Chungbuk 28119, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial
Received 11 September 2017 target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless
Accepted 15 September 2017 of the organism, are composed of homo-tetramers, except for two structures: Bacillus cereus and
Available online xxx
Staphylococcus aureus FabI (bcFabI and saFabI, respectively), which have been reported as dimers.
However, the reason for the existence of the dimeric form in these organisms and the biological meaning
Keywords:
of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal
Bacillus anthracis FabI
structure of a dimeric form of Bacillus anthracis FabI (baFabI) and the crystal structures of tetrameric
Dimeric form
Tetrameric form
forms of baFabI in the apo state and in complex with NADþ and with NADþ-triclosan, at 1.7 Å, 1.85 Å,
Dimer-tetramer transition 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that baFabI with a His6-tag at its C-terminus
exists as a dimer, whereas untagged-baFabI exists as a tetramer. The His6-tag may block the dimer-
tetramer transition, since baFabI has relatively short-length amino acids (255LG256) after the 310-helix
h7 compared to those of FabI of other organisms. The dimeric form of baFabI is catalytically inactive,
because the a-helix a5 occupies the NADH-binding site. During the process of dimer-tetramer transition,
this a5 helix rotates about 55 toward the tetramer interface and the active site is established. Therefore,
tetramerization of baFabI is required for cofactor binding and catalytic activity.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction
FabI is the final and rate-limiting step enzyme in the Fatty Acid
Synthase II (FAS II) system [1]. Owing to basic differences in the
fatty acids biosynthetic mechanisms between human (FAS I) and
bacteria (FAS II), the FAS II system is a useful target for anti-bacterial
drugs [2,3]. Many FabI inhibitors, such as isoniazid [4], diazabor-
anes [5], triclosan [6], and other small-molecule inhibitors [7e9],
have been developed as anti-bacterial agents. Clinical trials with
the FabI inhibitors CG400549 [10e12] and AFN-1252 [13,14] have
shown that they possess the safety, efficacy, and potency for the
treatment of staphylococcal infections.
The FabI structures reported to date in the Protein Data Bank
(PDB), regardless of the organism, are all composed of homo-
tetramers except for two structures: Bacillus cereus FabI (bcFabI)
* Corresponding author.
E-mail address: chahong@korea.ac.kr (K.Y. Hwang).
(PDB ID: 3OJE) [15] and Staphylococcus aureus FabI (saFabI) (PDB ID:
3GNS and 3GNT) [16], which are composed of dimers. Many cyto-
plasmic and membrane proteins form homo-oligomeric complexes
in cells and play roles in the regulation of cellular processes, such as
gene expression, activity of enzymes, ion channels, receptors, and
cell-cell adhesion [17]. Formation of homo-oligomers can also
provide sites for allosteric regulation and generate new ligand-
binding sites at the subunit interface(s) to increase specificity and
diversity [17,18]. Hence, one question arises: what is the underlying
biological significance of dimeric and tetrameric forms of FabI? To
answer the question, Bacillus anthracis FabI C-terminally fused to
His6-tag (baFabI-His) or lysozyme (baFabI-T4L) were engineered to
block dimer-tetramer transition and artificially maintain the dimer
form, respectively. Although the dimeric form of B. cereus FabI
(bcFabI), which is 100% identical to baFabI, is present in PDB, many
parts of the protein were disordered due to low resolution, 3.07 Å
[16]. Herein, we report the high-resolution crystal structure of the
dimeric form of baFabI and crystal structures of the tetrameric

http://dx.doi.org/10.1016/j.bbrc.2017.09.084
0006-291X/© 2017 Elsevier Inc. All rights reserved.

Please cite this article in press as: H.T. Kim, et al., Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis,
Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.09.084
2 H.T. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
forms of baFabI in the apo-state and in complex with NADþ and
with NADþ-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively.
Our data clearly revealed that conformational changes during
dimer-tetramer transition result in a rearrangement of the active
site into a catalytically competent conformation.
2. Materials and methods
2.1. Cloning and purification of Bacillus anthracis FabI
The full length B. anthracis FabI (baFabI) gene was cloned into
the BamHI/SacI sites of the expression vector, the modified pET21b
vector (Novagen). Escherichia coli BL21 (DE3) competent cells
transformed with the full-length baFabI construct were grown and
induced with 0.5 mM IPTG, for 15 h at 18  C. Cells were, then,
resuspended in buffer A [50 mM Tris pH 7.5, 200 mM NaCl, 5 mM b-
mercaptoethanol, 5% (w/v) glycerol, 1 mM PMSF]. The cell lysate
was cleared by centrifugation at 12,000 rpm, for 1 h, and the su-
pernatant loaded onto a Ni column (HisTrap HP 5 mL, GE Health-
care). The baFabI protein was eluted with buffer B (buffer A without
PMSF) containing 500 mM imidazole. Fractions containing baFabI
were pooled and incubated overnight with TEV protease. The
sample was purified using a heparin column (Hitrap Heparin HP
5 mL, GE Healthcare) and eluted using a linear 0e500 mM NaCl
gradient. Following a second Ni column to remove the His6-tag and
uncut protein, baFabI was further purified using a gel filtration
column (HiLoad 16/60 superdex 200 prep grade 120 mL, GE
Healthcare) and buffer C [20 mM Tris pH 7.5, 150 mM NaCl, 10% (w/
v) glycerol].
To produce baFabI-His, the baFabI gene was cloned into the
BamHI/SacI sites of the modified pET21b vector with a His6-tag at
the C-terminus. Transformation of competent cells, induction of
protein expression, and preparation of bacterial lysate was per-
formed as described above. Elution of baFabI-His was performed
with buffer B containing 500 mM imidazole, and the protein was
further purified using gel filtration and buffer C.
The baFabI-T4L gene was cloned into the BamHI/SacI/NotI sites of
the modified pET21b vector. The baFabI-T4L protein was eluted
with buffer B plus 500 mM imidazole. After pooling the fractions
containing baFabI-T4L and incubating the pooled sample overnight
with TEV protease, baFabI-T4L was further purified using gel
filtration and buffer C.
2.2. Enzymatic assay
The enzymatic activity of purified baFabI proteins was deter-
mined in 100 mM sodium acetate buffer (pH 6.5) with 4% glycerol
in the presence of 400 mM 2-butenoyl coenzyme A (Sigma-Aldrich)
and 200 mM NADH. The reaction was initiated by the addition of
FabI, which was used at various concentrations, at 30  C. Changes in
NADH absorbance were monitored for 1 h at 340 nm.
2.3. Multi-angle light scattering (MALS) analysis
MALS experiments were performed during size-exclusion
chromatography on a TSK-Gel G3000SWXL column (Tosoh) with
a MALS detector (DAWN HELEOS-II, Wyatt Technologies) and a
differential refractive-index detector (Optilab T-rEX, Wyatt Tech-
nologies). The weight-average molar masses were calculated from
the elution data, using ASTRA 6 software (Wyatt Technologies).
2.4. Crystallization, data collection, and structure determination
Purified baFabI was incubated for 3 h, at 4  C, with a 3-fold molar
excess of NADþ and triclosan. Cocrystallization experiments were
Please cite this article in press as: H.T. Kim, et al., Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis,
Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.09.084
set up using the hanging drop vapor diffusion method. Crystals of
baFabI in complex with NADþ and with NADþ and triclosan were
prepared in 0.1 M Na-acetate pH 4.5, 2 M ammonium sulfate.
Crystals of apo-baFabI were prepared in 0.1 M MES pH 6.0, 20e45%
(w/v) pentaerythritol proproxyllyate (5/4 PO/OH), 0.2 M NaCl.
Crystals of baFabI-His were prepared in 0.1 M Tris-HCl pH 8.5, 30%
PEG4K, 0.2 M MgCl2. Diffraction data of flash-frozen crystals were
collected on 5C beamline at Pohang Accelerator Laboratory. Data
were processed and scaled using HKL2000 [19]. All structures were
solved by molecular replacement using Phaser [20] with baFabI
(2QIO) as the initial search model. The model was manually con-
structed using COOT [21] and refined with PHENIX [22]. Data
collection and refinement statistics are provided in Table S1. The
coordinates and structure factors of a dimeric form of baFabI and
tetrameric forms of baFabI in the apo state and in complex with
NADþ and with NADþ-triclosan have been deposited in the PDB
with the codes 5YCX, 5YCV, 5YCR, and 5YCS, respectively.
3. Results and discussion
3.1. Crystal structures of dimeric and tetrameric forms of apo-
baFabI
Only three crystal structures of FabI from two organisms,
B. cereus (PDB ID: 3OJE) and S. aureus (PDB ID: 3GNS and 3GNT), in
PDB have been reported as dimeric forms [15,16]. The structures
have two common features: 1) they have relatively short-length
amino acids (255LG256 in bcFabI and 255IK256 in saFabI) at the C-
terminus after a highly conserved 310-helix h7 (Fig. 1A) and 2) they
have a His6-tag at the C-terminus. Therefore, we hypothesized that
the reason for these proteins to exist as dimers might be because
the His6-tag blocks the dimer-tetramer transition. However, in the
case of other organisms, including E. coli and Mycobacterium
tuberculosis, the structures of FabI exist as tetramers despite the
presence of a His6-tag at the C-terminus (Fig. 1B). Comparing with
baFabI or saFabI, E. coli FabI (ecFabI) has the additional C-terminal
residues 254AAMNELELK262 after the 310-helix h7 (Fig. 1A). The
formation of the ecFabI tetramer does not appear to be affected by
the His6-tag, since the residues are differently oriented, as shown in
Fig. 1C and D [23], and could act as a linker. To confirm our hy-
pothesis that tetramerization of baFabI is blocked by the His6-tag at
its C-terminus, we solved the structures of apo-baFabI with (baFabI-
His) and without the C-terminal His6-tag (baFabI-wt) at 1.7 and
1.85 Å, respectively. baFabI-wt existed as a tetramer (Fig. 2A),
whereas baFabI-His existed as a dimer with the crystallographic
two-fold symmetry-related monomer, molecule A’ (Fig. 2D). When
both structures were superimposed, steric clashes occurred be-
tween the C-terminal regions of both proteins: the additional C-
terminal residues þ1LEHHþ4 of molecule A0 in baFabI-His and the C-
terminal residues 255LG256 of molecule D in baFabI-wt (Fig. 2AeC).
Therefore, the His6-tag at the C-terminus blocks the dimer-
tetramer transition in baFabI-His.
In the dimeric form, the substrate-binding loop (SBL, 197e203),
the second substrate-binding loop (SBL-2, 99e111), and the active
site loop (ASL, 150e160), which play a key role in catalytic activity,
were disordered (Fig. 2E). The R111-S121 region was unfolded or
disordered in the dimeric forms of baFabI and saFabI reported in
previous studies [15,16], whereas it formed an a-helical confor-
mation in our dimeric structure, similar to that of tetrameric forms
of baFabI. However, compared to tetrameric forms, the a-helix was
rotated about 55 toward the NADH-binding site (Fig. 2F). As a
result, NADH is unable to bind to the baFabI-His dimer, because its
binding site is occupied by the a-helix a5 and parts of SBL-2 (I94-
N98) (Fig. 2F). This is consistent with the result of the enzymatic
activity (Fig. 3A). Therefore, our data show that the dimeric form of
 H.T. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6 3

Fig. 1. Sequence alignment and comparison of structures. (A) Sequence alignment of B. anthracis FabI, S. aureus FabI, and E. coli FabI. The residues highlighted in red boxes are
strictly conserved, while the residues in blue boxes are relatively conserved. The regions that are disordered in the dimer form (baFabI-His) are denoted by a dashed line. Secondary
structures of the ternary complex (baFabI-TCS) in this study are depicted above the sequence. The black box indicates the C-terminal residues after the highly conserved 310-helix h7.
This figure was prepared with ESPript [26]. (B) Superimposition of the homo-tetrameric form of baFabI on that of ecFabI (gray). The red box indicates the C-terminus of each
molecule. The three perpendicular two-fold noncrystallographic symmetry axes P, Q, and R are indicated. The interfaces spanned by axes P and R (Q and R) are referred to as the PR-
interface (QR-interface) [24]. (C) Top view of the C-terminal regions. The C-terminus of ecFabI is colored in blue. C-termini of baFabI are colored yellow and cyan. (D) Front view of
the C-terminal regions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

baFabI-His is inactive. a tetramer in solution [15]. Schiebel et al. show that the dimer and
the tetramer of saFabI are formed in a pH-dependent manner [24].
Thus, the oligomeric state, such as dimer or tetramer, might change
3.2. baFabI C-terminally fused to T4-lysozyme is a dimer and is
under variable conditions, such as pH, concentration, and
inactive
temperature.
In the baFabI-His structure, the His6-tag at the C-terminus does
To determine whether baFabI exists as a dimer or tetramer in
not form a secondary structure. Thus, the tag can be flexible. We
solution, we used size-exclusion chromatography coupled to multi-
hypothesized that the dimer or the tetramer might be controlled by
angle light scattering (SEC/MALS). SEC/MALS results indicate that
the His6-tag position: if the His6-tag is in a similar position as the C-
baFabI-wt and baFabI-His exist as tetramers, with molecular
terminus of E. coli FabI (Fig. 1C and D), baFabI-His might exist as a
weights of 102 kDa and 129 kDa, respectively (Fig. 3B and C). These
tetramer. To completely exclude the possibility, baFabI fused to T4
results are not consistent with our structural and enzymatic assay
lysozyme at the C-terminus (baFabI-T4L) was engineered to block
results. In previous studies, gel filtration and non-denaturing gel
dimer-tetramer transition and artificially maintain the dimer form.
electrophoresis suggested that, in solution, baFabI is a dimer [16].
baFabI-T4L was shown to be catalytically inactive (Fig. 3A). SEC/
However, although in crystals saFabI existed as a dimer, it existed as

Please cite this article in press as: H.T. Kim, et al., Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis,
Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.09.084
4 H.T. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6

Fig. 2. Crystal structure of baFabI-His. (A) Superimposition of baFabI-His on baFabI-wt (orange). The blue box indicates the C-terminus of each molecule. (B) Top view of the C-
terminal regions. (C) Front view of the C-terminal regions. (D) Holo-dimeric structure of baFabI-His. (E) Structure of the baFabI-His monomer. (F) Superimposition of baFabI-His
(gray) on baFabI in complex with NADþ (magenta). NADþ is represented by a sphere. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Fig. 3. Enzymatic activity and MALS data. (A) Enzymatic activity of baFabI-wt, baFabI-His, and baFabI-T4L. (BeD) MALS analysis of baFabI-wt (B), baFabI-His (C), and baFabI-T4L
(D). Elution of the protein from the SEC column was detected by UV (green), MALS (red), and RI (refractive index; blue), which was fitted to calculate the molecular mass (black line).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

MALS result shows that baFabI-T4L exists as a dimer in solution
(Fig. 3D). The dimeric form can be mediated by T4-lysozyme or
baFabI. However, there are no reports on T4-lysozyme existing as a
dimer in crystals and in solution [25]. Therefore, the dimeric form
of baFabI-T4L is mediated by baFabI. The dimeric form of baFabI-T4L
would be similar to that of baFabI-His.
3.3. Crystal structures of tetrameric baFabI in different states: apo,
NADþ-, and NADþ-triclosan-bound
We solved the structures of apo-baFabI (baFabI-wt), NADþ-
bound baFabI (baFabI-NAD), and NADH-triclosan-baFabI complex
(baFabI-TSC) without the His6-tag at the C-terminus. All structures

Please cite this article in press as: H.T. Kim, et al., Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis,
Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.09.084
 H.T. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6 5

Fig. 4. The process of dimer-tetramer transition. (A) Two dimers interact to form a tetramer in which the dimmers are related by a two-fold symmerty. (B) and (C) show front and
top views of the red box in (A). (D) Tetrameric form of baFabI. (E) and (F) show the front and top views of the red box in (D). The red and blue boxes show the QR interface
established by the 6-helix bundle (a5, a6, and a7 in molecules A and A0 ). The a5 helix, ASL, SBL, and SBL-2 are colored green, blue, yellow, and magenta, respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

existed as tetramers, like orthologous FabIs. When we compared
the structure of baFabI-wt with that of baFabI-NAD and of baFabI-
TSC, the r.m.s.d. values were 0.218 Å (for 250 Ca atom pairs) and
0.295 Å (for 256 Ca atom pairs), respectively. No significant struc-
tural differences were observed except for SBL (Fig. S1A). In the apo
form of baFabI-wt, this loop did not form any secondary structure
and was shifted ~14 Å away from the active site compared with
baFabI-TSC (Fig. S1B). However, SBL was disordered in baFabI-NAD
(Fig. S1C). In baFabI-TSC, 196LSAKG200, part of SBL, formed 310-helix
h5 and interacted with the 2, 4-dichlorophenyl ring of triclosan
(Fig. S1D). Therefore, triclosan binding induced the conformational
rearrangement from the open to the closed conformation, which
was similar to that of E. coli and S. aureus FabIs [6,15,24].
3.4. Tetramerization of baFabI is required for biological activity
Although baFabI and saFabI, in the absence of cofactor and in-
hibitor, were reported to be present as dimers in crystals [15,16],
the R111-S121 region was unfolded or disordered due to low res-
olution. In our dimeric form, this region is well-defined as a a-helix
(a5) (Figs. S2A and B). Dimer-tetramer transition is processed by
the interactions of a 6-helix bundle comprising a5, a6, and a7. The
tetramerization process, via the helix bundle, is as follows: two
dimeric molecules (molecule A:A0 ) interact to form a tetramer in
which the dimers are related by a two-fold symmetry (Fig. 4A). ASL,
which is disordered in the dimer, is rearranged by the helix-helix
a7-a70 interaction (Fig. 4B and C). Interestingly, the a-helix a5,
which occupies the active site, rotates approximately 55 toward
the QR interface, leading to its interaction with the a5 of the other
monomer (a5-a50 interaction) (Fig. 4DeF). During the process, SBL-
2, which is disordered in the dimeric form, is becomes ordered. As a
result of the interactions, the full tetramer interface and the active
site are established. Thus, the formation of the tetramer generates
the NADH-binding site.
Schiebel et al. showed that binding of NADPþ and triclosan to
dimeric saFabI induces simultaneous active site formation and
tetramerization [24]. However, this does not seem to occur in
baFabI. The dimeric form of baFabI is catalytically inactive, because
the a-helix a5 occupies the NADH-binding site. Therefore, tetra-
merization of baFabI is required for cofactor binding and catalytic
activity.
Conflict of interest
None to declare.
Financial support
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Acknowledgments
We thank the staff at the PLS 5C, 7A beamline for the assistance
in using their excellent facilities and help with X-ray data
collection.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.bbrc.2017.09.084.

Please cite this article in press as: H.T. Kim, et al., Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis,
Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.09.084
6 H.T. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
Transparency document
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