12 clu
3 tr
4 e
5 Gw
6
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9 cine,
10 ng T
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1 4
a r t i c l e i n f o
15 Article history:
16 Received 5 April 2011
17 Available online xxxx
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a b s t r a c t
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53 two alternatively spliced isoforms: secretory CLU (sCLU) and nu-
54 clear CLU (nCLU). As a heterodimer composed of a and b chains,
55 sCLU is secreted or resides inside cells by uptake of extracellular
56 CLU. On the other hand, nCLU lacks an endoplasmic reticulum
57 (ER)-targeting leader peptide and exists as non-glycosylated pre-
58 cursor nCLU (pnCLU) in the cytosol and as glycosylated form in
59 the nucleus [7].
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72ing the pro-apoptotic function of nCLU remains to be determined.
73The Bcl-2 family proteins are critical checkpoints of mitochon-
74drial apoptosis because they regulate the permeability of outer
75mitochondrial membrane and cytochrome c release from mito-
76chondria [16,17]. On the basis of the modular structure of Bcl-2
77homology (BH) domains, this family of proteins is divided into
78anti-apoptotic and pro-apoptotic subfamilies. Anti-apoptotic Bcl-
792 family members (e.g. Bcl-2, Bcl-XL, Mcl-1, Bcl-w and A1) have
80four conserved BH domains named BH1–BH4, whereas pro-apop-
81totic Bcl-2 family members have three domains named BH1–BH3
82(e.g. Bak, Bax and Bok) or only one domain named BH3 (BH3-only
⇑ Corresponding authors. Fax: +82 42 860 4598 (S.-W. Chi), +82 42 350 6814
(G.-S. Yi).
Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx
Contents lists availab
al
.e l
YBBRC 26599 No. of Pages 7, Model 5G
25 April 2011E-mail addresses: gsyi@kaist.ac.kr (G.-S. Yi), swchi@kribb.re.kr (S.-W. Chi).1. Introduction
Clusterin (CLU), also called apolipoprotein J or testosterone-re-
pressed prostate message 2 (TRPM-2), is a ubiquitously expressed
glycoprotein that is involved in a variety of cellular functions such
as apoptosis regulation, cell cycle regulation, DNA repair, lipid
transportation and complement inhibition [1,2]. CLU has drawn
attention as a target of novel cancer therapy because it is overex-
pressed in prostate and breast cancers and associated with cancer
promotion and metastasis [3–5]. Blocking its expression makes
cancer cells sensitive to apoptosis induced by chemotherapeutic
drugs [5,6]. Based on its cellular location, CLU is classified into
The role of CLU in apoptosis is complicated and differs depend-
ing on the cellular context [8–10]. The pleiotropic function of CLU
may arise from dynamic interactions of the different isoforms of
CLU with a variety of binding partners such as Ku70 and Bax
[11,12]. It has been reported that sCLU and intracellular CLU play
anti-apoptotic roles. Recently, Zhang et al. showed that intracellu-
lar CLU inhibits apoptosis by interacting with conformation-al-
tered Bax in mitochondria [12]. On the contrary, nCLU acts as a
pro-apoptotic signal, inhibiting cell growth and survival [9,13].
Overexpression of nCLU results in apoptosis and cell cycle arrest
by inhibiting nuclear factor-jB (NF-jB)-dependent Bcl-XL expres-
sion [14,15]. However, the precise molecular mechanism underly-Keywords:
Clusterin
Apoptosis
BH3 domain
NMR
Bcl-2 family protein0006-291X/$ - see front matter  2011 Published by
doi:10.1016/j.bbrc.2011.04.054
Please cite this article in press as: D.-H. Lee et al
NMR spectroscopy, Biochem. Biophys. Res. ComClusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers.
Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molec-
ular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this
study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence
analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apop-
totic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift pertur-
bation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding
grooves in both Bcl-XL and Bcl-2. A structural model of the Bcl-XL/nCLU BH3 peptide complex reveals that
the binding mode is remarkably similar to those of other Bcl-XL/BH3 peptide complexes. In addition,
mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in
the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-XL. Taken altogether, our results
suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific inter-
actions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.
 2011 Published by Elsevier Inc.Interaction of a putative BH3 domain of
family proteins as revealed by NMR spec
Dong-Hwa Lee a, Ji-Hyang Ha a, Yul Kimb, Kwang-He
Ho Sup Yoon e, Sung Goo Park a, Byoung Chul Park a,
aMedical Proteomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea
bDepartment of Bio and Brain Engineering, KAIST, Daejeon 305-701, Republic of Korea
cDepartment of Physiology, Institute of Health Sciences, Republic of Korea
dDepartment of Anatomy and Neurobiology, Biomedical Center (BK21), College of Medi
eDivision of Structural and Computational Biology, School of Biological Sciences, Nanya
Biochemical and Biophysic
journal homepage: wwwElsevier Inc.
., Interaction of a putative BH3 d
mun. (2011), doi:10.1016/j.bbrsterin with anti-apoptotic Bcl-2
oscopy
Bae a, Jae Yong Park c, Wan Sung Choi d,
an-Su Yi b,⇑, Seung-Wook Chi a,⇑
Gyeongsang National University, Jinju, Gyeongnam 660-751, Republic of Korea
echnological University, 60 Nanyang Drive, Singapore 637511, Singapore
le at ScienceDirect
Research Communications
sevier .com/locate /ybbrcomain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by
c.2011.04.054

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85 cell death and survival through direct interactions between anti-
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97 Bak and Bax. However, sensitizers/derepressors (e.g. Bad and Noxa)
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l Res
YBBRC 26599 No. of Pages 7, Model 5G
25 April 2011can only interact with anti-apoptotic Bcl-2 family proteins.
The BH3-only protein family is an expanding family of proteins
that includes Bid, Bim, Bad, Noxa, Puma, Bmf, Bik and Hrk [18]. The
number of members has continues to increase with the recent dis-
covery of new members such as apolipoprotein I6 [19] and Beclin-
1 [20]. The protein–protein interactions of BH3-only family pro-
teins are mainly mediated by the BH3 domain. It contains the 7-
residue core sequence of LXXXGDE (X, any amino acid) or the
13-residue consensus sequence motif ‘‘/1RXX/2XX/3R0DZ/4C’’
(/1–/4, hydrophobic residues; R and R0, small residues; Z, an
acidic residue; C, a hydrophilic residue) [21]. The previous finding
of the interaction of CLU with a Bcl-2 family protein Bax [12] sug-
gests the possibility that CLU may have a BH3 motif. However, the
direct molecular evidences of this putative BH3 motif in CLU and
its binding specificity with Bcl-2 family proteins have not been
studied yet.
In this study, we searched a putative BH3 motif in nCLU using
the known BH3 motif information and identified a highly con-
served BH3 motif in C-terminal coiled coil (CC2) region of nCLU.
To examine whether the putative BH3 motif of nCLU acts as an
authentic BH3 motif of BH3-only protein, we performed nuclear
magnetic resonance (NMR) binding experiments of the corre-
sponding BH3 domain in nCLU with anti-apoptotic Bcl-2 family
proteins Bcl-XL and Bcl-2. The structural model of the complex
based on the chemical shift perturbation data showed that the
nCLU BH3 motif binds to the pro-apoptotic BH3 binding grooves
of anti-apoptotic Bcl-XL and Bcl-2, retaining the specific interaction
pattern of typical BH3 motif with Bcl-2 family proteins. Taken to-
gether with mutagenesis data, our results provide a direct evidence
of the residue specific interaction between the putative BH3 motif
in nCLU and anti-apoptotic Bcl-2 family proteins, suggesting that
nCLU can sequester the anti-apoptotic Bcl-2 family proteins by
BH3 motif specific molecular interaction for the apoptosis process.
2. Materials and methods
2.1. Identification of BH3 motif on nCLU sequence
Total 58 known BH3 motif sequences were collected from PRO-
SITE database [22] and literatures [17,18,20]. The sequences were
aligned with MUSCLE [23] and a hidden Markov model (HMM)
for conserved BH3 motif sequences was generated by using
HMMER [24]. To identify the putative BH3 motif, human nCLU se-
quence was scanned with HMM profile of BH3 motif.
2.2. Preparation of proteins and peptidesapoptotic and pro-apoptotic family members [16]. The pro-apopto-
tic effector Bcl-2 family proteins Bax and Bak form oligomers to
generate membrane pores, leading to mitochondrial outer mem-
brane permeabilization (MOMP) and cytochrome c release from
mitochondria. However, anti-apoptotic Bcl-2 family proteins bind
and sequester the pro-apoptotic effectors Bax and Bak, preventing
them from forming membrane pores. In particular, BH3-only pro-
teins are subdivided into ‘‘direct activators’’ and ‘‘sensitizers/dere-
pressors’’. The direct activators (e.g. Bid and Bim) that interact with
anti-apoptotic Bcl-2 family proteins as well as pro-apoptotic effec-
tors can directly induce apoptosis through the oligomerization ofproteins). In response to various physiological and pathological
stimuli, the Bcl-2 family of proteins maintains the balance between
2 D.-H. Lee et al. / Biochemical and BiophysicaA recombinant human nCLU-BH3 domain construct corre-
sponding to residues 308–343 and its mutant domains (L323E
and D328K) were expressed using the pGEX-2T vector in
Please cite this article in press as: D.-H. Lee et al., Interaction of a putative BH3 d
NMR spectroscopy, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bbr316–336) peptide was calculated using the NMR data-driven dock-
ing program HADDOCK 2.0 [29]. Using the software Discovery Stu-
dio (Accelrys), the initial structural model of the nCLU-BH3 (316–
336) peptide was built from the structure of the Bad-BH3 peptide
in complex with Bcl-XL (PDB accession code: 1G5J) [30] by the
homology modeling. Ambiguous interaction restraints (AIRs) for
Bcl-XL and nCLU-BH3 (316–336) peptide were defined based on
NMR chemical shift perturbation data. The active site residues of
Bcl-XL were defined as solvent-accessible amino acids with signifi-
cant chemical shift perturbations. A total of 2000 rigid-bodydocking
solutions were obtained and the best 200 structures were selected
for semi-flexible refinement and finally refined in explicit water.
3. Results and discussion
3.1. nCLU interacts with anti-apoptotic Bcl-2 family proteins via a
conserved BH3 domain
In the sequence analysis of nCLU with hidden Markov model
(HMM) profile of known BH3 motif sequences, we identified resi-
dues 316–336 in the CC2 region of nCLU as a putative BH3 motif
sharing the highly conserved consensus region around Leu323
and Asp328 (Fig. 1). Based on the result that nCLU contains a highly
conserved BH3 motif, we postulated that nCLU may bind to anti-
apoptotic Bcl-2 family proteins via the BH3 motif. To test our
hypothesis, we examined, using NMR spectroscopy, whether the
putative BH3 domain can actually bind to anti-apoptotic Bcl-2
family proteins. For this, we expressed and purified an nCLU do-
main containing the putative BH3 motif (residues 308–343, herein
referred to as nCLU-BH3) and performed NMR binding experi-
ments of Bcl-XL and Bcl-2 with the nCLU-BH3 domain. Fig. 2A
shows 2D 1H–15N HSQC spectra of 15N-labeled Bcl-XL and Bcl-2
in the absence or presence of the nCLU-BH3 domain. Upon binding
to the nCLU-BH3 domain, a significant portion of the crosspeaks of
Bcl-XL and Bcl-2 disappeared or changed their positions, indicating
the physical direct binding of the nCLU-BH3 domain to Bcl-XL. The
disappearance of the crosspeaks of Bcl-XL and Bcl-2 in the 2D
1 15Escherichia coli BL21(DE3) RIL cells and then purified using a gluta-
thione agarose column and a Sephacryl S200 FPLC column. The
15N-labeled human Bcl-XLDloopDTM and Bcl-2 chimera constructs
with truncation in the transmembrane segment and the flexible
loop region were expressed and purified as previously reported
[25,26] for NMR experiments.
2.3. NMR spectroscopy
All NMR data were acquired on a Bruker Avance II 900 spec-
trometer equipped with a cryogenic probe. The 2D 1H–15N HSQC
spectra of 15N-labeled Bcl-2 and Bcl-XL were obtained at 25 C in
the absence or presence of wild-type and mutant nCLU-BH3 do-
mains. NMR samples of 95% H2O/5% D2O were prepared in
20 mM sodium phosphate (pH 6.5), 50 mM NaCl, 1 mM DTT for
Bcl-XL and 20 mM Tris–HCl (pH 7.8) and 5 mM DTT for Bcl-2. For
chemical shift perturbation experiments with the anti-apoptotic
Bcl-2 family proteins, the wild-type and the mutant nCLU-BH3 do-
mains were added to 15N-labeled Bcl-2 or Bcl-XL during titration.
All NMR data were processed and analyzed using an NMRPipe/
NMRDraw [27] and SPARKY software [28].
2.4. Structure calculation
earch Communications xxx (2011) xxx–xxx198H– N HSQC spectra may be attributed to severe line broadening
199due to intermediate exchange on the NMR time scale. Our results
200demonstrated that nCLU directly interacts with anti-apoptotic
omain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by
c.2011.04.054