Novel dimerization mode of the human Bcl-2 family protein Bak,
a mitochondrial apoptosis regulator
Hongfei Wanga, Chie Takemotoa, Ryogo Akasakaa, Tomomi Uchikubo-Kamoa, Seiichiro Kishishitaa,
Kazutaka Murayamaa,b, Takaho Teradaa, Lirong Chenc, Zhi-Jie Liuc, Bi-Cheng Wangc, Sumio Suganod,
Akiko Tanakaa, Makoto Inouea, Takanori Kigawaa, Mikako Shirouzua, Shigeyuki Yokoyamaa,e,*
aSystems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
bGraduate School of Biomedical Engineering, Tohoku University, Sendai 980-8575, Japan
cDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30603, USA
dDepartment of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 113-0033, Japan
eDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
a r t i c l ei n f o
Received 29 August 2008
Received in revised form 1 December 2008
Accepted 8 December 2008
Available online 24 December 2008
Bcl-2 family protein
a b s t r a c t
Interactions of Bcl-2 family proteins play a regulatory role in mitochondrial apoptosis. The pro-apoptotic
protein Bak resides in the outer mitochondrial membrane, and the formation of Bak homo- or heterodi-
mers is involved in the regulation of apoptosis. The previously reported structure of the human Bak pro-
tein (residues Glu16–Gly186) revealed that a zinc ion was coordinated with two pairs of Asp160 and
His164 residues from the symmetry-related molecules. This zinc-dependent homodimer was regarded
as an anti-apoptotic dimer. In the present study, we determined the crystal structure of the human
Bak residues Ser23–Asn185 at 2.5 Å, and found a distinct type of homodimerization through Cys166
disulfide bridging between the symmetry-related molecules. In the two modes of homodimerization,
the molecular interfaces are completely different. In the membrane-targeted model of the S–S bridged
dimer, the BH3 motifs are too close to the membrane to interact directly with the anti-apoptotic relatives,
such as Bcl-xL. Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode
under oxidized conditions.
? 2008 Elsevier Inc. All rights reserved.
Commitment of cells to apoptosis is mainly governed by pro-
tein–protein interactions among the Bcl-2 protein family mem-
bers. Structural and functional studies have identified the
importance of conserved Bcl-2 homology motifs (BH1, BH2, BH3,
and BH4) in many family members (Cory and Adams, 2002; Danial
and Korsmeyer, 2004; Petros et al., 2004; Delft and Huang, 2006).
The Bcl-2 protein family consists of three subfamilies, which are
characterized by the number of BH motifs (Bouillet et al. 2002;
Newmeyer and Ferguson-Miller, 2003; Willis et al., 2007). The hu-
man anti-apoptotic proteins include Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1,
and Bcl-B, which possess three or four of the BH motifs. The pro-
apoptotic BH3-only members, which act as sensors of specific
types of cellular stress, include Bid, Bim, Puma, Bad, Noxa, Bmf,
Hrk, and Bik (Willis andAdams, 2005). The pro-apoptotic multi-do-
main proteins, which function downstream of the former two
groups and possess BH1–3, are represented by Bax, Bak, and Bok
(Lindsten et al., 2000).
Either Bax or Bak is required for apoptosis; they appear to be
largely redundant in function. Whereas the loss of either single
gene has few effects in most cells and tissues, the absence of both
proteins blocks apoptosis in many cell types (Wei et al., 2001). A
notable biochemical difference between Bax and Bak is that, in
healthy cells, Bax is primarily cytosolic or loosely associated with
mitochondria, whereas Bak is an integral membrane protein on
the cytosolic faces of the mitochondrion and the endoplasmic
reticulum. Some models for the regulation of Bak by its anti-
apoptotic relatives have been proposed, as follows. In healthy cells,
Bcl-xLand Mcl-1 can bind to Bak, which is presumably a ‘primed’
conformer with its BH3 exposed, while in apoptosis-induced cells,
a BH3-only protein displaces Bak from the anti-apoptotic heterodi-
mer (Adams and Cory, 2007). The free Bak then forms an oligomer
(of unknown structure) that elicits the permeabilization of the
mitochondrial outer membrane and the release of cytochrome
c (Green, 2005).
Structuralstudieshave beenperformedon Bcl-2family proteins,
including both the anti-apoptotic (Bcl-xL, Bcl-2, Bcl-w, Mcl-1) and
pro-apoptotic (Bax, Bid, Bak) members (Petros et al., 2004; O’Neill
1047-8477/$ - see front matter ? 2008 Elsevier Inc. All rights reserved.
* Corresponding author. Address: Systems and Structural Biology Center, Yoko-
hama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045,
Japan. Fax: +81 45 503 9195.
E-mail address: email@example.com (S. Yokoyama).
Journal of Structural Biology 166 (2009) 32–37
Contents lists available at ScienceDirect
Journal of Structural Biology
journal homepage: www.elsevier.com/locate/yjsbi
et al., 2006; Moldoveanu et al., 2006; Czabotar et al., 2007). All of
them adopt helical structures with a prominent hydrophobic
groove, which serves as the binding site for the BH3 motif of its
counterpart. The solution structure of Bax revealed that its own
C-terminal helix occupies the hydrophobic groove, representing
the structure in the cytosol of healthy cells (Suzuki et al., 2000).
The structure of Bcl-xLin complex with the BH3 peptides of Bak
provided information about the heterodimerization, which is
important for anti-apoptosis (Sattler et al., 1997). Recently, the
crystal structure of calpain-proteolyzed Bak, in the space group
C2, revealed that Bak formed a zinc-dependent homodimer, in
which the BH3 motif is buried against the anti-apoptotic relative
in the membrane-targeted form (Moldoveanu et al., 2006). The
addition of zinc ion inhibits the release of cytochrome c from the
mitochondrial membrane, suggesting that the zinc-dependent
homodimer is involved in anti-apoptosis.
On the other hand, the structural information concerning apop-
tosis is poorly understood. A recent cell biological study suggested
a pro-apoptotic model of Bak homodimer formation via interac-
tions between the BH3 motif and the hydrophobic groove (Dewson
et al., 2008). Meanwhile, we describe here the crystal structure of
human Bak in the space group P6522, in which Bak forms an S–S
bridged homodimer. A similar structure was also deposited in
the PDB, with the accession code 2JCN by the Structural Genomics
Consortium. Furthermore, we investigated the states of Bak in
solution by dynamic light scattering (DLS) and mass spectrometry
(MALDI-TOF-MS). A recent study showed that mitochondria-med-
iated apoptosis by diallyl trisulfide in human prostate cancer cells
is associated with the generation of reactive oxygen species (Kim
et al., 2007). Taken together, the crystal structure of the S–S
bridged Bak provides more information for understanding the pos-
sible pathway of apoptosis.
2. Materials and methods
2.1. Protein expression, purification, and crystallization
The DNA encoding residues Ser23–Asn185 of human Bak (or
Bcl-2 homologous antagonist killer, NP-001179 in the NCBI data-
base) was cloned into the expression vector pCR2.1 TOPO (Invitro-
gen), as a fusion with an N-terminal His-tag and a TEV protease
cleavage site. The selenomethionine (SeMet)-substituted Bak
(Ser23–Asn185) was synthesized by the Escherichia coli cell-free
system (Kigawa et al., 2004). The reaction solution was centrifuged
at 16,000g at 4 ?C for 20 min. The supernatant was loaded onto a
HisTrap column (5 ml), equilibrated with 20 mM Tris–HCl buffer
(pH 8.0), containing 1 M NaCl and 20 mM imidazole. After washing
the column with the buffer, the His-tagged protein was eluted with
20 mM Tris–HCl buffer (pH 8.0), containing 500 mM NaCl and
500 mM imidazole. The sample buffer was exchanged to 20 mM
Tris–HCl buffer (pH 8.0), containing 1 M NaCl and 20 mM imidaz-
ole, with a HiPrep 26/10 desalting column. The His-tag was cleaved
by 100 ll of TEV protease (4 mg/ml) at 30 ?C for 1 h. To remove the
uncleaved protein, the reaction solution (14 ml) was loaded onto a
HisTrap column (5 ml), as described above. The flow-through frac-
tions were collected and desalted on a HiPrep 26/10 desalting
column with 20 mM Tris–HCl buffer (pH 8.0), containing 5 mM
2-mercaptoethanol (2ME). The pooled fraction (30 ml) was loaded
onto a HiTrap Q column (5 ml), equilibrated with 20 mM Tris buf-
fer (pH 8.0), containing 5 mM 2ME, 10 mM NaCl, and protease
inhibitors (Complete Mini EDTA-free tablet, Roche), and the Bak
protein was eluted with a linear gradient of 10 mM to 1 M NaCl. Fi-
nally, the purified protein fraction (6 ml) was concentrated to
1.5 ml with an Amicon Ultra-15 filter (5000 MWCO, Millipore),
and wasloadedona HiLoad16/60 Superdex75column,
equilibrated with 20 mM Tris–HCl buffer (pH 8.0), containing
150 mM NaCl and 2 mM dithiothreitol (DTT). The purified protein
was concentrated to 11.1 mg/ml (450 ll) for crystallization. All
chromatography materials were purchased from GE Healthcare
The crystals were obtained in drop composed of 2 ll protein
solution and 2 ll reservoir solution, by the hanging drop vapor dif-
fusion technique at 20 ?C. The reservoir solution contained 1.8 M
ammonium sulfate and 3% v/v 2-propanol (pH 5.2).
2.2. Data collection, structure determination, and refinement
Synchrotron diffraction data were collected to 2.5 Å resolution
from a single crystal on beamline 22-ID at the Advanced Photon
Source (APS), using a MAR mosaic 300 CCD detector with a wave-
length of 0.97105 Å at 100 K. The diffraction data were processed
and scaled with HKL-2000 (Otwinowski and Minor, 1997). The
structure was solved by the Se-SAD method. The coordinates for
all three Se atoms were found in the 2.5 Å dataset, using the pro-
gram SOLVE (Terwilliger and Berendzen, 1999), and the initial
model containing 120 of the 166 residues was built using the pro-
gram RESOLVE (Terwilliger, 2000). The models were rebuilt manu-
ally using O (Jones et al., 1991) and were refined with CNS (Brunger
et al., 1998). The structures were refined to an R-factor of 21.4%
(Rfree= 24.8%) at 2.5 Å resolution. Structural alignments were done
with the programs DEJAVU and LSQMAN (Kleywegt and Jones,
1997) and LSQKAB (Kabsch, 1976). The protein secondary structure
was defined by the DSSP algorithm (Kabsch and Sander, 1983). The
quality of the model was inspected by the program PROCHECK
(Laskowski et al., 1993). Graphic figures were created using the
programs Pymol and Grasp (Nicholls et al., 1991; DeLano, 2002).
2.3. DLS and MALDI-TOF-MS determination
The monomeric and dimeric protein samples were purified by
size-exclusion chromatography (Superdex-75 10/30) just prior to
the experiments. The monomer was prepared by incubating the
protein solution at 35 ?C for 4 h in the presence of 10 mM 2ME.
The S–S bridged homodimer was prepared by dialysis against
20 mM Tris–HCl buffer (pH 7.5), containing 150 mM NaCl and
without DTT, at 4 ?C for 24 h. Firstly, the monomeric and dimeric
states of Bak in solution were examined by a dynamic light scatter-
ing (DLS) experiment, using a Zetasizer Nano S System (Malvern
Instruments Ltd.). The concentration of the protein solution was
2.0 mg/ml, in 20 mM Tris–HCl buffer (pH 8.0) containing 150 mM
NaCl. Several measurements were taken at different temperatures
and were analyzed using the Dispersion Technology Software (ver-
sion 4.20). The multiple narrow-mode analysis was utilized for the
calculations. The states of the purified proteins were monitored by
SDS–PAGE, and the molecular masses were determined by mass
spectrometry (MALDI-TOF-MS, Applied Biosystems Voyager Sys-
tem), using myoglobin as a standard.
3.1. Overall structure
The crystal structure of Bak (Ser23–Asn185), in the space group
P6522, was determined by the Se-SAD method and was refined to
an R-factor of 21.4% (Rfree= 24.8%) at 2.5 Å resolution (Table 1).
The structure has been deposited in the Protein Data Bank (PDB),
with the accession code 2YV6. There is a single molecule in the
asymmetric unit of the crystal. The final refined model contains
residues 23–48 and 52–184 of the Bak protein. Residues 49–51
and residue 185 were not defined in the electron density map,
H. Wang et al./Journal of Structural Biology 166 (2009) 32–37
and they are assumed to be disordered. A stereo ribbon diagram of
the protein is shown in Fig. 1a. The overall structure of the protein
consists of eight a-helices. A structure-based sequence alignment
of Bak and its Bcl-2 relatives is shown in Fig. 1b. Two central heli-
ces (a5 and a6) form the core of the protein. These two helices are
predominately hydrophobic, and are flanked on one side by a3 and
a4, and on the other side by a1 and a2. The signature ‘‘NWGR” se-
quence, which is highly conserved among Bcl-2 family members,
directly precedes a5. The BH3 domain is located between a2 and
a3. In the crystal, Bak forms a dimer by the disulfide bonding of
Cys166 with the symmetry-related molecule, although the protein
was monomeric before crystallization.
Two structures of the human Bak protein have been deposited in
the PDB, with the accession codes 2IMS (Moldoveanu et al., 2006)
and 2JCN. Although the amino acid lengths are slightly different,
the three overall structures of the Bak protein are very similar, as
shown in Fig. 1c. Cys166 forms a disulfide bridge with that of the
study) and 2JCN. On the other hand, a zinc ion was coordinated in
2IMS. It is notable that the crystallization conditions had different
pH values: pH 7.5 for 2JCN, pH 5.2 for 2YV6 (this study), and pH
4.6 for 2IMS (Moldoveanu et al., 2006). However, the crystal struc-
ture of Bak was not affected over the pH range of 4.6–7.5. Similarly,
no structural change of the other pro-apoptotic protein, Bax, in
solution was observed over the pH range of 6–8, in a series of 2D
(1H–15N) nuclear magnetic resonance spectra (Suzuki et al., 2000).
The exposed hydrophobic binding groove, formed mainly by a3,
a4, and a5, is the important site for the design of Bcl-2 family pro-
tein inhibitors, to develop potential anti-cancer therapeutics
(Oltersdorf et al., 2005). A sulfate ion resides in the vicinity of
the hydrophobic groove, and Trp125, Trp170, Asn124, and
Arg174 are located around the sulfate ion. These residues are
somewhat conserved among Bak, Bax, Bcl-2, and Bcl-xL. Arg174
of Bak is altered to Asn in Bcl-xL, which contributes to a stronger
positive charge for the sulfate-ion binding. Similar to the structure
Fig. 1. (a) Stereo ribbon diagram of Bak with the secondary structure elements labeled. The BH3 motif is colored cyan. The sulfate ion is shown as a yellow stick model. (b) A
structure-based sequence alignment of Bak (2YV6), Bax (1F16), Bcl-xL(1MAZ), and Bcl-2 (1G5M) was performed with the programs DaliLite (Holm and Park, 2000) and
CLUSTAL-X (Thompson et al., 1997). The secondary structural elements, determined on the basis of the crystal structure of Bak, are indicated with coils for alpha and 310
helices at the top of the alignment. BH motifs conserved in Bcl-2 relatives are labeled at the bottom of the alignment. The identical amino acid residues are shown in white
letters highlighted in red, while the similar residues are shown in red letters. (c) Overall and superpostioned structures of Bak. 2YV6 (this study), 2JCN, and 2IMS are green,
pale-green, and lemon, respectively. The zinc ion and the sulfate ion are shown as an orange sphere and a yellow stick model, respectively.
Summary of data collection and refinement statistics.
Unit-cell dimensions (Å, ?)
a = 61.824, b = 61.824, c = 137.482
a = b = 90, c = 120
No. of observations
No. of unique reflections
RMSD bonds (Å)/angles (?)
Dihedrals (?)/improper (?)
Ramachandran plot, residues in
Most favored regions (%)
Additional allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
aNumbers in parentheses represent values in the highest resolution shell (2.50–
bRsym=P|Ij? hIi|/PIj, where Ijis the observed integrated intensity, hIi is the
mation is over all observed reflections.
cRcryst=P||Fobs| ? |Fcalc||/P|Fobs|, Fobs and Fcalc are observed and calculated
dRfreecalculated with randomly selected reflections (5%).
average integrated intensity obtained from multiple measurements, and the sum-
structure factor amplitudes, respectively.
H. Wang et al./Journal of Structural Biology 166 (2009) 32–37
of 2IMS, the side-chain stacking of Arg88 and Tyr89 on this pocket
generates an occluded conformation of Bak, which interferes with
the binding of the BH3 peptide.
3.2. Homodimer formation
A cartoon representation of the S–S bonded Bak homodimer
structure is shown in Fig. 2. The Fo–Fcmap, contoured at 3r, clearly
shows the density corresponding to the S–S bridge of Cys166. The
crystal packing is different from that of 2IMS, which forms a zinc-
dependent homodimer (Moldoveanu et al., 2006). The zinc ion is
coordinated in a tetrahedral geometry by two pairs of adjacent res-
idues, Asp160 and His164. The ‘‘shoulder by shoulder” (Form I) di-
mer is formed in 2IMS, and the ‘‘head to head” dimer (Form II) is
formed in 2YV6 and 2JCN. To compare these two forms, the buried
surface areas between the subunits were calculated with the pro-
tein–protein interaction analysis server (Jones and Thornton,
1995). The buried surface area of Form I is 538 Å2, and that of Form
II is 510 Å2. In addition, hydrophobic and hydrophilic interactions
were observed at the interface in both forms. Thus, although puri-
fied Bak in solution is a monomer, Bak possibly forms homodimers
with different contact surfaces on the mitochondrial membrane. It
is unlikely that the disulfide bridge is formed under the reducing
conditions in normal cells, even if the two Cys166 residues in the
Form II dimer are close enough. Consequently, the disulfide bond
formation in Form II may further stabilize the dimer under special,
oxidative conditions, as discussed below.
3.3. Biophysical analyses of Bak in solution
To investigate the predominant species and state of Bak in solu-
tion, dynamic light scattering (DLS) and MALDI-MS analyses were
performed. The monomer and the S–S bridged dimer were
prepared just before these experiments (Fig. 3a), as described in
In the DLS measurements, a particle-size dispersity of 11.4%
was observed for the freshly purified Bak, and the molecular mass
was estimated to be 22.5 kDa, which corresponds to the mono-
meric state. After the sample was stored at 4 ?C for six months, a
particle-size dispersity of 16.7% was observed, and the molecular
mass was estimated to be 43.5 kDa, which is consistent with the
dimeric state. These results suggest that Bak became oxidized,
even in the presence of 2 mM DTT at 4 ?C. The oxidized dimer is
stable over the pH range of 5–8, as confirmed by SDS–PAGE, and
at temperatures from 10 to 50 ?C, as confirmed by DLS analyses
(data not shown).
The disulfide bond of the S–S dimer was reduced by an incuba-
tion with 10 mM 2ME. The protein solution contained the mono-
mer and the S–S bridged dimer, as detected by SDS–PAGE with
or without 2ME treatment (Fig. 3b). The molecular mass of each
purified fraction of the monomer and the dimer was measured
by MALDI-TOF-MS. The estimated molecular masses of the mono-
mer and the dimer are 19,152 and 38,304 kDa, respectively. The
MALDI-TOF-MS spectra generated molecular masses of 19,123.0
and 38,358.6 kDa for the monomer and the dimer, respectively
All of the above results clearly showed that the Bak monomer
was oxidized to the S–S bridged dimer, and that the dimer is stable
In healthy cells, the anti-apoptotic relative Bcl-xLbinds to the
BH3 motif of Bak on the mitochondrial membrane, while in apop-
totic cells, Bak molecules dimerize and form oligomers (Adams and
Cory, 2007). As shown in Fig. 4a and b, we modeled a membrane-
targeted Bak homodimer in Form II with the C-terminus embedded
in the mitochondrial membrane (Fig. 4a), as the Zn2+-mediated
anti-apoptotic homodimer in Form I was modeled previously
(Moldoveanu et al., 2006) (Fig. 4b). Based on the NMR structure
of the Bcl-xLcomplexed with the BH3 peptide from Bak (PDB ID:
1BXL) (Sattler et al., 1997), the interface of Bak with Bcl-xLinvolves
BH3 residues buried in the monomeric structure of Bak (Fig. 4c). To
interact with Bcl-xL, the interface residues are required to be ex-
Fig. 3. (a) Purification of the Bak monomer (solid line) and dimer (dotted line) by
Superdex-75 10/30 chromatography. (b) Confirmation of the S–S bridged dimer by
SDS–PAGE. The loading solution only for the lane 2 sample contained 2ME. Lanes 1,
2: Bak stored for six months. Lanes 3, 4: Bak dimer (3) and monomer (4) purified by
Superdex-75. (c) MALDI-TOF-MS spectra of the purified Bak as the monomer
(upper) and the dimer (lower).
Fig. 2. Structure of the Bak homodimer formed by the S–S bond. The symmetry-
related molecule is shown in blue. Each helix is colored the same as in Fig. 1a. The
lower panel shows the Fo–Fc map (magenta) around the S–S bond of Cys166,
contoured at 3r.
H. Wang et al./Journal of Structural Biology 166 (2009) 32–37
posed with a large conformational change of Bak, which would re-
sult in the BH3-everted conformation proposed previously (Willis
and Adams, 2005; Dewson et al., 2008). The BH3 motif in Form I
may be exposed for binding with Bcl-xL(Fig. 4b), and has been pro-
posed to represent the anti-apoptotic mode (Moldoveanu et al.,
2006). In contrast, the BH3 motif in Form II faces the membrane
closely and is unlikely to bind Bcl-xL(Fig. 4a). Therefore, the Form
II dimer structure may represent one of the pro-apoptotic modes,
which should be prohibited by Bcl-xL. On the other hand, another
homodimer model generated by interactions between the BH3 mo-
tif and the hydrophobic groove of Bak has been proposed (Dewson
et al., 2008).
It is unlikely that the disulfide bridge is formed under the
reducing conditions in normal cells, even if the two Cys166 resi-
dues in the Form II dimer are close enough. Actually, the disulfide
bridge formation is not required for the pro-apoptotic activity of
Bak, as the C166S mutant possesses pro-apoptotic activity in
mouse embryonic fibroblast cells (Moldoveanu et al., 2006; Dew-
son et al., 2008). On the other hand, even for androgen-dependent
prostate cancer cells (LNCaP), in which androgen prevents the cells
from undergoing apoptosis, diallyl trisulfide is still able to induce
the generation of reactive oxygen species (ROS) and reduce the
Bcl-2/Bcl-xLlevel, thereby inducing apoptosis dependent on Bax/
Bak (Kim et al., 2007). An antioxidant removes ROS impairs the
Bax/Bak-dependent apoptosis, indicating the importance of ROS
for the Bax/Bak-dependent apoptosis. Under these conditions, the
absence of Bcl-2/Bcl-xLprobably favors the formation of the Form
II dimer of Bak on the mitochondrial membrane. Furthermore, the
disulfide bridge may easily be formed under the oxidative condi-
tions due to ROS, and may thereby stabilize the Form II dimer (or
the pro-apoptotic dimer) of Bak in the ROS- and Bak/Bax-depen-
In the early stage of the cell death process, Bak reportedly forms
large complex clusters with Bax (Nechushtan et al., 2001), which
requires its BH3 motif for its pro-apoptotic activity and oligomer-
ization (Oltvai et al., 1993; Wang et al., 1998; Willis et al., 2005). A
detailed study revealed that the BH3 and BH1 motifs are responsi-
ble for the Bax homodimerization (George et al., 2007). Bax is the
closest homologue of Bak in function, amino acid sequence, and
structure. The Bax structure lacking the C-terminal helix can be
superposed on that of Bak, with a backbone atomic root-mean-
square deviation of 1.9 Å for the Ca atoms of residues 27–39, 70–
80, and 111–184 (Fig. 4d). The conformation and the surface
charge of the BH3 motifs are similar to each other, while the hydro-
phobic pocket around BH3 exhibits an open conformation in Bax.
In addition, the residues coordinating the zinc ion in Bak, Asp160
and His 164, are conserved as Asp142 and Glu146 in Bax. Although
Cys166 of Bak is not conserved in Bax, Cys126, between a5 and a6,
is exposed in the Bax structure. These similarities suggest that Bax
could form a ‘‘foot to foot” S–S bridged dimer. Overall, the structure
of the Bak homodimer provides a possible structural basis for the
oligomerization of Bak and Bax in the pro-apoptotic mode.
Fig. 4. Cartoon representation of the membrane-targeted Bak homodimer in Form II (a) and Form I (b). The C-terminal putative trans-membrane region (I188-S212), the
C-terminal helix a8 and the BH3 motif are colored orange, blue, and cyan, respectively. The connecting linker GNGP187 is shown as a dotted line, and the hydrophobic groove
is indicated by a gray, ellipsoidal region. (c) Cartoon representation of the NMR structures of Bcl-xL(yellow cartoon) and the Bak BH3 peptide complex (1BXL; left), and the
crystal structure of Bak (2YV6; right). Residues in the BH3 motif that interact with the groove of Bcl-xLare shown as stick models. Each helix of Bak is colored the same as in
Fig. 1a. (d) Superposition of Bak (2YV6 in green) and Bax (1F16 in light blue). The C-terminal helix of Bax (1F16) was removed for clarity. The residues involved in dimerization
are shown as stick models, and the BH3 domains are shown in light yellow (Bak) and pink (Bax), respectively. Structural alignments were performed with the programs
DEJAVU, LSQMAN and LSQKAB.
H. Wang et al./Journal of Structural Biology 166 (2009) 32–37
Acknowledgments Download full-text
We thank T. Yabuki, M. Aoki, E. Seki, T. Matsuda, Y. Tomo, and S.
Watanabe for constructing the expression vector with the rapid
screening system by NMR methods, and Y. Kinoshita for sample
preparation. We also thank T. Takagi and M. Shida for helpful dis-
cussions, and A. Ishii and T. Nakayama for manuscript preparation.
This work was supported by the RIKEN Structural Genomics/Pro-
teomics Initiative (RSGI), the National Project on Protein Structural
and Functional Analysis, Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan.
Adams, J.M., Cory, S., 2007. The Bcl-2 apoptotic switch in cancer development and
therapy. Oncogene 26, 1324–1337.
Bouillet, P., Purton, J.F., Godfrey, D.I., Zhang, L.C., Coultas, L., Puthalakath, H.,
Pellegrini, M., Cory, S., Adams, J.M., Strasser, A., 2002. BH3-only Bcl-2 family
member Bim is required for apoptosis of autoreactive thymocytes. Nature 415,
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve,
R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M.,
Simonson, T., Warren, G.L., 1998. Crystallography & NMR system: a new
software suite for macromolecular structure determination. Acta Crystallogr. D
Cory, S., Adams, J.M., 2002. The Bcl2 family: regulators of the cellular life-or-death
switch. Nat. Rev. Cancer 2, 647–656.
Czabotar, P.E., Lee, E.F., van Delft, M.F., Day, C.L., Smith, B.J., Huang, D.C., Fairlie,
W.D., Hinds, M.G., Colman, P.M., 2007. Structural insights into the degradation
of Mcl-1 induced by BH3 domains. Proc. Natl. Acad. Sci. USA 104, 6217–6222.
Danial, N.N., Korsmeyer, S.J., 2004. Cell death: critical control points. Cell 116, 205–
DeLano, W.L., 2002. The PyMOL Molecular Graphics System. DeLano Scientific, San
Carlos, CA, USA.
Delft, M.F., Huang, D.C.S., 2006. How the Bcl-2 family of proteins interact to regulate
apoptosis. Cell Res. 16, 203–213.
Dewson, G., Kratina, T., Sim, H., Puthalakath, H., Adams, J.M., Colman, P.M., Kluck,
R.M., 2008. Totrigger apoptosis,
homodimerizes via BH3: groove interactions. Mol. Cell 30, 369–380.
George, N.M., Evans, J.J.D., Luo, X., 2007. A three-helix homo-oligomerization
domain containing BH3 and BH1 is responsible for the apoptotic activity of Bax.
Genes Dev. 21, 1937–1948.
Green, D.R., 2005. Apoptotic pathways: ten minutes to dead. Cell 121, 671–674.
Holm, L., Park, J., 2000. DaliLite workbench for protein structure comparison.
Bioinformatics 16, 566–567.
Jones, S., Thornton, J.M., 1995. Protein–protein interactions: a review of protein
dimer structures. Prog. Biophys. Mol. Biol. 63, 31–65.
Jones, T.A., Zou, J.Y., Cowan, S.W., Kjeldgaard, J., 1991. Improved methods for
building protein models in electron density maps and the location of errors in
these models. Acta Crystallogr. A 47, 110–119.
Kabsch, W., 1976. A solution for the best rotation to relate two sets of vectors. Acta
Crystallogr. A 32, 922–923.
Kabsch, W., Sander, C., 1983. Dictionary of protein secondary structure: pattern
recognition of hydrogen-bonded and geometrical features. Biopolymers 22,
Kigawa, T., Yabuki, T., Matsuda, N., Matsuda, T., Nakajima, R., Tanaka, A., Yokoyama,
S., 2004. Preparation of Escherichia coli cell extract for highly productive cell-
free protein expression. J. Struct. Funct. Genomics 5, 63–68.
Kim, Y.A., Xiao, D., Xiao, H., Powolny, A.A., Lew, K.L., Reilly, M.L., Zeng, Y., Wang, Z.,
Singh, S.V., 2007. Mitochondria-mediated apoptosis by diallyl trisulfide in
human prostate cancer cells is associated with generation of reactive oxygen
species and regulated by Bax/Bak. Mol. Cancer Ther. 6, 1599–1609.
Kleywegt, G.J., Jones, T.A., 1997. Detecting folding motifs and similarities in protein
structures. Methods Enzymol. 277, 525–545.
Bak exposesitsBH3 domainand
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK: a
program to check the stereochemical quality of protein structures. J. Appl. Cryst.
Lindsten, T., Ross, A.J., King, A., Zong, W., Rathmell, J.C., Shiels, H.A., Ulrich, E.,
Waymire, K.G., Mahar, P., Frauwirth, K., Chen, Y., Wei, M., Eng, V.M., Adelman,
D.M., Simon, M.C., Ma, A., Golden, J.A., Evan, G., Korsmeyer, S.J., MacGregor, G.R.,
Thompson, C.B., 2000. The combined functions of proapoptotic Bcl-2 family
members Bak and Bax are essential for normal development of multiple tissues.
Mol. Cell 6, 1389–1399.
Moldoveanu, T., Liu, Q., Tocilj, A., Watson, M., Shore, G., Gehring, K., 2006. The X-ray
structure of a BAK homodimer reveals an inhibitory zinc binding site. Mol. Cell
Nechushtan, A., Smith, C.L., Lamensdorf, I., Yoon, S.H., Youle, R.J., 2001. Bax and Bak
coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell
Biol. 15, 1265–1276.
Newmeyer, D.D., Ferguson-Miller, S., 2003. Mitochondria: releasing power for life
and unleashing the machineries of death. Cell 112, 481–490.
Nicholls, A., Sharp, K.A., Honig, B, 1991. Protein folding and association: insights
from the interfacial and thermodynamic properties of hydrocarbons. Proteins
Oltersdorf, T., Elmore, S.W., Shoemaker, A.R., Armstrong, R.C., Augeri, D.J., Belli, B.A.,
Bruncko, M., Deckwerth, T.L., Dinges, J., Hajduk, P.J., Joseph, M.K., Kitada, S.,
Korsmeyer, S.J., Kunzer, A.R., Letai, A., Li, C., Mitten, M.J., Nettesheim, D.G., Ng,
S.C., Nimmer, P.M., O’Connor, J.M., Oleksijew, A., Petros, A.M., Reed, J.C., Shen,
W., Tahir, S.K., Thompson, C.B., Tomaselli, K.J., Wang, B., Wendt, M.D., Zhang, H.,
Fesik, S.W., Rosenberg, S.H., 2005. An inhibitor of Bcl-2 family proteins induces
regression of solid tumours. Nature 435, 677–681.
Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with
a conserved homolog, Bax, that accelerates programmed cell death. Cell 74,
O’Neill, J.W., Manion, M.K., Maguire, B., Hockenbery, D.M., 2006. BCL-XL
dimerization by three-dimensional domain swapping. J. Mol. Biol. 356, 367–
Otwinowski, Z., Minor, W., 1997. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol. 276, 307–326.
Petros, A.M., Olejniczak, E.T., Fesik, S.W., 2004. Structural biology of the Bcl-2 family
of proteins. Biochim. Biophys. Acta 1644, 83–94.
Sattler, M., Liang, H., Nettesheim, D., Meadows, R.P., Harlan, J.E., Eberstadt, M., Yoon,
H.S., Shuker, S.B., Chang, B.S., Minn, A.J., Thompson, C.B., Fesik, S.W., 1997.
Structure of Bcl-xL–Bak peptide complex: recognition between regulators of
apoptosis. Science 275, 983–986.
Suzuki, M., Youle, R.J., Tjandra, N., 2000. Structure of Bax: coregulation of dimer
formation and intracellular localization. Cell 103, 645–654.
Terwilliger, T.C., 2000. Maximum-likelihood density modification. Acta Crystallogr.
D 56, 965–972.
Terwilliger, T.C., Berendzen, J., 1999. Automated MAD and MIR structure solution.
Acta Crystallogr. D 55, 849–861.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The
CLUSTAL-X windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.
Wang, K., Gross, A., Waksman, G., Korsmeyer, S.J., 1998. Mutagenesis of the BH3
domain of BAX identifies residues critical for dimerization and killing. Mol. Cell.
Biol. 18, 6083–6089.
Wei, M.C., Zong, W.X., Cheng, E.H., Lindsten, T., Panoutsakopoulou, V., Ross, A.J.,
Roth, K.A., MacGregor, G.R., Thompson, C.B., Korsmeyer, S.J., 2001. Proapoptotic
BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.
Science 292, 727–730.
Willis, S.N., Adams, J.M., 2005. Life in the balance: how BH3-only proteins induce
apoptosis. Curr. Opin. Cell Biol. 17, 617–625.
Willis, S.N., Chen, L., Dewson, G., Wei, A., Naik, E., Fletcher, J.I., Adams, J.M., Huang,
D.C., 2005. Pro-apoptotic Bak is sequestered by Mc1-1 and Bcl-xL, but not Bcl-2,
until displaced by BH3-only proteins. Genes Dev. 19, 1294–1305.
Willis, S.N., Fletcher, J.I., Kaufmann, T., van Delft, M.F., Chen, L., Czabotar, P.E., Ierino,
H., Lee, E.F., Fairlie, W.D., Bouillet, P., Strasser, A., Kluck, R.M., Adams, J.M.,
Huang, D.C.S., 2007. Apoptosis initiated when BH3 ligands engage multiple Bcl-
2 homologs, not Bax or Bak. Science 315, 856–859.
H. Wang et al./Journal of Structural Biology 166 (2009) 32–37