Ku B, Woo JS, Liang C, Lee KH, Hong HS, E X et al.. Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68. PLoS Pathog 4: e25

Oregon Health and Science University, United States of America
PLoS Pathogens (Impact Factor: 7.56). 03/2008; 4(2):e25. DOI: 10.1371/journal.ppat.0040025
Source: PubMed
ABSTRACT
Author Summary

In higher animals, defective or surplus cells are removed by a process known as apoptosis. On the other hand, defective or damaged cellular components are removed by a process known as autophagy. These two destructive processes are indispensable for the survival and development of an organism. While apoptosis is known as a central host defense mechanism that removes virus-infected cells, the role of autophagy against viral infection has recently emerged. Many viruses express an armory of viral proteins that counteract cell death–mediated innate immune control. One such protein is a homologue of the cellular BCL-2 protein that suppresses apoptosis through inhibitory binding to apoptosis-promoting proteins. Murine γ-herpesvirus 68 also encodes a viral BCL-2, known as M11. In this study, we quantitatively measured the binding affinity of M11 for its potential cellular targets, including ten different proapoptotic proteins and the proautophagic protein Beclin1. We found that M11 neutralizes the proapoptotic proteins broadly rather than selectively to suppress apoptosis. Surprisingly, M11 bound to Beclin1 with the highest affinity, which correlated with its strong antiautophagic activity in cells. These data suggest that M11 suppresses not only apoptosis but also autophagy potently, which ultimately contributes to the viral chronic infection.

Full-text

Available from: Xiaofei Ee, Jun 19, 2014
Structural and Biochemical Bases for the
Inhibition of Autophagy and Apoptosis
by Viral BCL-2 of Murine c-Herpesvirus 68
Bonsu Ku
1[
, Jae-Sung Woo
1[
, Chengyu Liang
2
, Kwang-Hoon Lee
1
, Hyang-Suk Hong
1
, Xiaofei E
2
, Key-Sun Kim
3
,
Jae U. Jung
2
, Byung-Ha Oh
1*
1 Division of Molecular and Life Sciences, Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea, 2 Department of
Microbiology and Molecular Genetics and Tumor Virology Division, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts, United
States of America, 3 Biomedical Research Center, Korea Institute of Science and Technology, Seoul, Korea
All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of
infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2
proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine c-
herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides
derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the
BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the
1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more
efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the
upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively
suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a
similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the
virus.
Citation: Ku B, Woo JS, Liang C, Lee KH, Hong HS, et al. (2008) Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine c-
herpesvirus 68. PLoS Pathog 4(2): e25. doi:10.1371/journal.ppat.0040025
Introduction
Gammaherpesviruses are DNA viruses comprising a sub-
family of the Herpesviridae. These viruses, including Epstein-
Barr virus, Kaposi’s sarcoma-associated herpesvirus (KSHV)
and murine c-herpesvirus 68 (cHV68), are etiological agents
of lymphoid and epithelial tumors in human or animals [1,2].
All c-herpesviruses encode at least one homologue of the
cellular apoptosis inhibitor BCL-2, and expression of these
viral BCL-2 genes prevents cell death under various apopto-
sis-inducing conditions [3–6]. In particular, critical roles of
the BCL-2 homologue of cHV68 have been determined by in
vitro and in vivo studies in the pathogenesis of the cHV68
virus. The protein, known as and referred to as M11 here,
protected cells from undergoing apoptosis induced by a
variety of factors, such as dexamethasone treatment, c-ray
irradiation, CD3e ligation [7], tumor necrosis factor treat-
ment [8,9], Fas ligation [9], and Sindbis virus infection [10].
Furthermore, the protein contributed to latency establish-
ment [11] and was required for efficient reemergence from
latency as well as persistent replication during chronic
infection of the virus in immunocompromised mice lacking
interferon-c [12]. These data indicate that removal of virus-
infected cells by cell death is a central host defense
mechanism against viral infection, and viral BCL-2 proteins
play a crucial role in the course of viral replication by
inhibiting the death of host cells [1,13,14].
The BCL-2 family proteins, which are commonly known as
positive or negative regulators of apoptosis, are characterized
as containing up to four conserved stretches of amino acids,
known as BCL-2 homology (BH) domains [15,16]. BH3-only
proteins, a group of proapoptotic BCL-2 family including
BIM, BAD, PUMA and Noxa, sense prodeath signals and
ultimately activate the downstream proapoptotic members
BAX and BAK [17,18]. Activated BAX and BAK cause
mitochondrial dysfunction and lead to the release of
proapoptogenic molecules, such as cytochrome c [19,20].
The interactions between such proapoptotic BCL-2 family
members and the antiapoptotic members, such as BCL-2 and
BCL-X
L
, are the crucial events in controlling or promoting
Editor: Klaus Fru
¨
h, Oregon Health and Science University, United States of America
Received August 17, 2007; Accepted December 21, 2007; Published February 1,
2008
Copyright: Ó 2008 Ku et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: cHV68, murine c-herpesvirus 68; BAD, BCL-2-associated death
promoter; BAK, BCL-2-antagonist/killer 1; BAX, BCL-2-associated X protein; BCL-2, B-
cell lymphoma-2; BH, BCL-2 homology; BID, BH3-interacting domain death agonist;
BIK, BCL-2-interacting killer; BIM, BCL-2-interacting mediator of cell death; BMF,
BCL-2-modifying factor; CCD, coiled-coil domain; CD, circular dichroism; ECD,
evolutionarily conserved domain; GFP, green fluorescent protein; GST, glutathione-
S-transferase; Hrk, harakiri; HT, hydrophobic tail; ITC, isothermal titration
calorimetry; KSHV, Kaposi’s sarcoma-associated herpesvirus; LC3, light chain 3 of
microtubule-associated protein 1; MCL-1, myeloid cell leukemia sequence 1;
PI(3)KCIII, class III phosphatidylinositol 3-kinase; PUMA, p53-upregulated mediator
of apoptosis; TFE, trifluoroethanol; UVRAG, UV irradiation resistance-associated
gene
* To whom correspondence should be addressed. E-mail: bhoh@postech.ac.kr
[ These authors contributed equally to this work.
PLoS Pathogens | www.plospathogens.org February 2008 | Volume 4 | Issue 2 | e250001
Page 1
apoptosis [15,16]. These interactions are mediated by the BH3
domain of the proapoptotic members that binds to a site
known as the BH3-binding groove in the antiapoptotic
members [21,22].
In addition to their critical roles in the regulation of
apoptosis, the B CL-2 family proteins have emerged as
regulators of autophagy, a catabolic process that plays crucial
roles in cell survival, tumor suppression, and innate immune
defense against intracellular pathogens by degrading cyto-
plasmic components through lysosomal pathway [23–25]. The
leading work was the identification of Beclin1 as a BCL-2-
interacting protein [26]. A series of subsequent studies
showed that Beclin1 promotes autophagy as a component
of a multiprotein complex containing class III phosphatidy-
linositol 3-kinase (PI(3)KCIII) and UV irradiation resistance-
associated gene (UVRAG) [27–29], and that BCL-2 negatively
regulates the autophagy-promoting activity of Beclin1 [30],
while the BH3-only protein BAD plays an autophagy-
stimulatory function by disrupting the interaction of BCL-2
or BCL-X
L
with Beclin1 [31]. While Beclin1 exhibits no
overall sequence homology with the BCL-2 family proteins,
the recently reported structure of BCL-X
L
in complex with a
Beclin1 peptide revealed the presence of a novel BH3 domain
in Beclin1 that binds to the BH3-binding groove of BCL-X
L
[32]. As observed with the cellular kin, expression of the viral
BCL-2 protein of KSHV or cHV68 significantly inhibits
autophagy in a Beclin1 binding-dependent manner [28,30],
suggesting that these two viral BCL-2 proteins may function
as autophagy inhibitors as well as apoptosis inhibitors.
In this study, we determined the structure of M11 in
complex with a 50-residue Beclin1 fragment containing its
BH3-like domain. Ensuing analyses revealed that M11 binds
Beclin1 significantly more tightly than cellular BCL-2 through
tighter hydrophobic interactions. Consistently, transiently
expressed M11 inhibited autophagosome formation more
efficiently than cellular BCL-2. We also quantified the
interactions of M11 with the BH3 peptides derived from
the apoptosis mediators BAX and BAK and the eight
upstream BH3-only proapoptotic molecules BAD, BIK, BIM,
BID, BMF, PUMA, Noxa and Hrk. The binding affinity of M11
was highest for Beclin1 and fairly high for BAK, BIM, Noxa,
BID, BMF and PUMA, but comparatively low for BAX and
Hrk. In the observed affinity profile, M11 is distinctively
different from cellular BCL-2 and also from M11L, a
virulence factor of Myxoma virus and a structural mimic of
BCL-2 that acts primarily by sequestering BAX and BAK [33].
These data suggest that M11 robustly inhibits the Beclin1-
dependent autophagy and broadly neutralizes the proapop-
totic BCL-2 family to subvert the host antiviral responses.
Results
Interaction of Beclin1 with M11
Mouse Beclin1 is composed of 448 amino acids. By
coexpression test, we found that mouse Beclin1 fragment
consisting of residues 101–150, which spans the structurally
defined BCL-2-binding region consisting of residues 105–125
(corresponding to residues 107–127 of human Beclin1 [32]),
formed a tight complex with M11 lacking the C-terminal
hydrophobic tail. The protein in complex with Beclin1(101–
150) was crystallized and its structure was determined to 2.3 A
˚
resolution (Table 1). Residues 106–124 of Beclin1 form an a-
helix and bind M11 at an extended hydrophobic surface cleft
corresponding to the BH3-binding groove of BCL-X
L
[7]
(Figure 1A). In the crystal, the N-terminal five and the C-
terminal 26 residues of the Beclin1(101–150) peptide were
Table 1. Data Collection and Structure Refinement Statistics
M11–Beclin1 BCL-X
L
–BAD
Space group P2
1
P6
5
Unit cell dimensions
a, b, c (A
˚
) 42.87, 53.60, 73.59 91.75, 91.75, 58.54
Wavelength (A
˚
) 1.0000 1.0000
Resolution (A
˚
) 30.0–2.3 30.0–2.2
R
sym
a
6.4 (16.2)
b
7.8 (28.2)
I/r(I) 25.2 (6.2) 11.0 (2.0)
Completeness (%) 94.0 (80.5) 89.3 (67.8)
Redundancy 4.9 2.5
Refinement
Resolution (A
˚
) 20.0–2.3 20.0–2.3
Number of reflections 13,447 11,244
R
work
c
/ R
free
20.4 / 24.7 21.9 / 24.8
Number of atoms
Protein 2,444 1,389
Water 59 38
R.m.s deviations
Bond lengths (A
˚
) 0.0065 0.0068
Bond angles (8) 1.0627 1.1953
Ramachandran plot (%)
Most favored region 90.4 93.3
Additionally allowed region 9.6 5.4
Generously allowed region 1.3
Average B-values (A
˚
2
)
Protein 28.3 (M11) 29.4 (BCL-X
L
)
Peptide 41.5 (Beclin1) 24.0 (BAD)
Water 27.3 27.5
a
R
sym
¼ R jI
obs
- I
avg
j / I
obs
, where I
obs
is the observed intensity of individual reflection and
I
avg
is average over symmetry equivalents.
b
The numbers in parentheses are statistics from the highest resolution shell.
c
R
work
¼ R jjF
o
j - jF
c
jj / R jF
o
j, where jF
o
j and jF
c
j are the observed and calculated structure
factor amplitudes, respectively. R
free
was calculated with 5% of the data.
doi:10.1371/journal.ppat.0040025.t001
PLoS Pathogens | www.plospathogens.org February 2008 | Volume 4 | Issue 2 | e250002
Bases for Inhibition of Host Cell Death by M11
Author Summary
In higher animals, defective or surplus cells are removed by a
process known as apoptosis. On the other hand, defective or
damaged cellular components are removed by a process known as
autophagy. These two destructive processes are indispensable for
the survival and development of an organism. While apoptosis is
known as a central host defense mechanism that removes virus-
infected cells, the role of autophagy against viral infection has
recently emerged. Many viruses express an armory of viral proteins
that counteract cell death–mediated innate immune control. One
such protein is a homologue of the cellular BCL-2 protein that
suppresses apoptosis through inhibitory binding to apoptosis-
promoting proteins. Murine c-herpesvirus 68 also encodes a viral
BCL-2, known as M11. In this study, we quantitatively measured the
binding affinity of M11 for its potential cellular targets, including ten
different proapoptotic proteins and the proautophagic protein
Beclin1. We found that M11 neutralizes the proapoptotic proteins
broadly rather than selectively to suppress apoptosis. Surprisingly,
M11 bound to Beclin1 with the highest affinity, which correlated
with its strong antiautophagic activity in cells. These data suggest
that M11 suppresses not only apoptosis but also autophagy
potently, which ultimately contributes to the viral chronic infection.
Page 2
disordered. The binding of Beclin1(101–150) induces a
conformational change of M11 to reshape the BH3-binding
groove (Figure 1B). Residues 53–55, a loop segment tailing
from a2 in free M11, form an additional helical turn of a2in
Beclin1(101–150)-bound M11 (Figure 1B). In addition, a3 and
the following segment undergo a significant conformational
transition that involves the translocation of several residues
by a distance of 4–10 A
˚
(Figure 1B).
In order to test whether the crystal structure reflects the
interaction of Beclin1 with M11 in solution and to determine
the strength of their interaction, we performed a quantitative
binding analysis using isothermal titration calorimetry (ITC)
(Figure 1C and Table S2). We employed a Beclin1 fragment
containing residues 101–267 (referred to as Beclin1(101–
267)), since this fragment was expressed as a soluble form in E.
col i while Beclin1(101–150) was not. This large Beclin1
fragment bound to M11 very tightly w ith an apparent
dissociation constant (K
D
) of 40 nM (Figure 1C). Similar
binding affinity (K
D
of 99 nM) was observed with a synthetic
Beclin1(101–125) peptide (Figure 1C). In contrast, a shorter
Beclin1 fragment composed of residues 101–116 exhibited no
sign of interaction with the protein (not shown). Unexpect-
edly, a synthetic Beclin1(106–125) peptide showed quite low
binding affinity (K
D
of 1.6 lM) for M11 (Figure 1C), suggesting
Figure 1. Structural and Binding Analyses of the M11–Beclin1(101–150) Complex
(A) Ribbon drawing (left) and surface presentation (right). M11 is in pink, while the Beclin1 helix is in green. The pink and green regions on the primary
sequence diagrams indicate the fragments of the M11 and Beclin1 used for the structure determination. ‘‘ HT’’ , ‘‘ CCD’’ and ‘‘ ECD’’ denote hydrophobic
tail, coiled-coil domain and evolutionarily conserved domain, respectively. Only 19 amino acids of Beclin1 exhibited well-defined electron density. A
surface presentation of M11 with the omission of the Beclin1 helix shows that the BH3-binding groove is predominantly hydrophobic. The surface
coloring scheme is as follows: olive for Val, Leu, Ile, Phe, Trp, Met, and Ala; yellow for Cys, Gly, Tyr, and Pro; gray for other amino acids.
(B) Large conformational change of M11 induced by the Beclin1 binding. Only the BH3-binding groove region of M11 is shown for clarity. Beclin1(101–
150)-bound M11 (pink) and free M11 (cyan) are superposed. The bound Beclin1 peptide is in green. Helix a3 of M11 undergoes a pronounced
conformational change. The arrows indicate the movements of the Ca atoms of Asp59 and Tyr60 in M11.
(C) ITC analysis. The measurements were carried out by titrating 0.1 mM of M11 into 5 lM of the indicated Beclin1 fragments. The K
D
values were
deduced from curve fittings of the integrated heat per mol of added ligand and summarized in the table.
(D) M11 interacts with endogenous Beclin1. NIH3T3 cells were transfected with HA-tagged M11 and whole cell lysates were used for
immunoprecipitation with anti-HA followed by immunoblotting with anti-Beclin1
doi:10.1371/journal.ppat.0040025.g001
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Bases for Inhibition of Host Cell Death by M11
Page 3
that residues 101–105 of Beclin1 constitute an important
piece in t he interaction of Beclin1 peptide with M11,
although these five residues were disordered in the crystal
and thus are not likely to interact with M11. It was previously
shown that residues 140–144 and 161–164 of a BAD peptide
contribute to the binding affinity by increasing the helical
propensity of the peptide rather than by interacting with
BCL-X
L
[34]. Similarly, a circular dichroism (CD) spectro-
scopic analysis showed that the Beclin1(101–125) peptide has
considerably higher helical contents (29.6%) compared with
the Beclin1(106–125) peptide (17.0%) in 30% trifluoroetha-
nol (TFE) solution (Figure S1). The data supports the idea that
residues 101–105 of Beclin1 promote the binding of the
Beclin1(101–125) peptide to M11 by increasing the helical
propensity of the following segment. Conclusively, M11 binds
Beclin1 with potently high affinity, and residues 101–125 of
Beclin1 compose the minimal region sufficient for binding to
M11. In a reflection of the observed potent interaction, we
could easily detect the interaction between transiently
expressed full-length M11 and endogenous Beclin1 in
NIH3T3 cells (Figure 1D).
Comparison with BCL-X
L
–BAD Complex
Cellular antiapoptotic BCL-2 family members share high
sequence homology in the BH1, BH2 and BH3 domains,
which compose the common and characteristic BH3-binding
groove [35]. At a glance, the intermolecular interaction
between M11 and Beclin1(101–150) resembled the interac-
tions between the BH3-binding groove of cellular antiapop-
totic BCL-2 relatives and a BH3-domain containing peptide
or fragment [21,22,36]. For a detailed structural comparison,
we used the crystal structure of BCL-X
L
in complex with BAD
that we have determined to 2.3 A
˚
resolution (Table 1), in
which 27 residues of BAD bound to BCL-X
L
as an extended a-
helix and all the rest of the residues were totally disordered. A
sequence alignment based on the structural comparison
showed that four out of five residues within proapoptotic
BH3 domains that are critical for their interactions with the
BH3-binding groove [21] are conserved as Leu110, Leu114,
Asp119 and Phe121 in Beclin1 (Figure 2A and 2B). The
remaining residue, which is isoleucine or methionine in the
BH3 domains, is substituted as Thr117 in Beclin1. These five
residues occupy spatially and chemically equivalent positions
at the BH3-binding groove of M11 as the corresponding
residues of BAD bound to BCL-X
L
(Figure 2A). Additional
structural comparison involving the BCL-X
L
–BAK, BCL-X
L
BIM and MCL-1–BIM complexes led to the same conclusion,
as the five residues are conserved in the BH3 domains of
BAD, BAK and BIM (Figure 2B) and they occu py the
equivalent positions at the BH3-binding groove of BCL-X
L
or MCL-1 (Figure S2). The side chain hydroxyl group of
Thr117 of Beclin1 is situated in a hydrophobic milieu, and
therefore this residue appeared to make an insignificant or
adverse contribution to the helix-groove interaction, in
contrast with isoleucine or methionine in the canonical
BH3 domains. Thr117 is conserved in the Beclin1 orthologues
of vertebrates, but not in those of lower organisms (Figure
2C). Threonine for this position might have been chosen to
tune the affinity of Beclin1 for cellular BCL-2 or BCL-X
L
at a
physiologically optimum level. Another noticeable difference
from the canonical BH3 domains is that the Beclin1 a-helix
has a hydrophobic patch composed of Val116, Leu120 and
Ile123 that are not shielded by the BH3-binding groove
(Figures 2D and S3), while those of other BH3 domains,
including that of BAD (Figures 2D and S3), are distinctively
amphipathic. The exposed hydrophobic residues of Beclin1
are identically or similarly conserved throughout species
(Figure 2C), suggesting that they may play an as yet unknown
important role. These structural and sequence comparisons
indicate that Beclin1 has an atypical BH3 domain charac-
terized by the threo nine substitution and the exposed
hydrophobic patch.
M11 Interacts with Beclin1 Much More Tightly and Inhibits
Autophagy More Potently than Cellular BCL-2
In contrast to the robust interaction between M11 and
Beclin1(101–267), we found that BCL-2 interacts with
Beclin1(101–267) weakly with a K
D
of 1.7 lM (Figure 3A),
which is similar to the K
D
value (1.1 lM) for the interaction
between BCL-X
L
and a Beclin1 peptide [32]. In order to
account for the huge difference in the binding affinity, we
compared our structure with the BCL-X
L
–Beclin1 peptide
structure [32]. Compared with 950 A
˚
2
interface of BCL-X
L
buried by 22 residues of Beclin1, the binding interface of M11
is smaller (860 A
˚
2
) and involves fewer Beclin1 residues (a total
of 16 residues). However, the binding surface of M11 renders
tighter hydrophobic interactions with Beclin1 compared with
that of BCL-X
L
(Figure 3B). For example, while Phe121 of
Beclin1 interacts with Ala93 of BCL-X
L
, it interacts with the
corresponding but bulkier residue Leu44 of M11 (Figure 3B).
Another notable difference is that the bound Beclin1 helix
interacts tightly with the a3 helix of M11, while it interacts
poorly with the corresponding region in BCL-X
L
(Figure 3B),
which consistently exhibits poor electron density (Figure S4)
and high temperature factors [32]. As a result of these and
other differences in the binding interactions, the M11–
Beclin1 helix makes 88 intermolecular carbon-carbon con-
tacts (distance , 4.2 A
˚
), while the BCL-X
L
–Beclin1 helix
makes 76 such contacts, indicating that the marked difference
in the binding affinity arises from the difference in the shape
complementarity, and thus the quality, of the hydrophobic
interactions.
To explore whether the marked difference in the binding
affinity of M11 and BCL-2/ BCL-X
L
for Beclin1(101–267)
indeed corre lates with their activity, we measured the
autophagy-inhibiting capacity of M11 and cellular BCL-2.
To quantify the level of autophagy, green fluorescent protein-
tagged light chain 3 of microtubule-associated protein 1
(GFP–LC3) was used to indicate the formation of autopha-
gosomes, which deliver cellular components to lysosomes for
degradation and recycling during autophagy. GFP–LC3, a
specific marker for autophagosome, moves from the peri-
nuclear region into autophagosomal membranes under
autophagy-promoting con ditions such as starvation and
rapamycin treatment [37,38]. In NIH3T3 mouse fibroblast
cells, transiently expressed M11 inhibited autophagosome
formation more efficiently than transiently expressed BCL-2,
as evident from the rate of GFP–LC3 positive cells carrying
autophagic vacuoles and the number of autophagosomes per
cell, while the expression level of M11 was much less than that
of BCL-2 (Figure 4A and 4B). The efficacy of M11 and BCL-2
was dose-dependent, as the ratio of autophagosome-carrying
cells decreased with the increase of the amount of vectors
used for transfection (Figure 4C). In these analyses,
PLoS Pathogens | www.plospathogens.org February 2008 | Volume 4 | Issue 2 | e250004
Bases for Inhibition of Host Cell Death by M11
Page 4
M11(AAA), the M11 mutant containing alanine substitutions
of three conserved residues (S85A, G86A and R87A) within
the BH3-binding groove [7] and barely able to bind Beclin1
[28], exhibited significantly reduced antiautophagic activity
compared with the wild-type protein (Figure 4A, 4B, and 4C),
suggesting that the Beclin1-binding capacity is essential for
the antiautophagic activity of M11. To further compare their
antiautophagic capacity, immunoblotting was also performed
with an antibody against LC3. LC3-II, a cleavage product
generated from the LC3 precursor (LC3-I), accumulates in
the autophagosomal membrane during autophagy and there-
fore is widely used as a specific marker for autophagy
processing [38,39]. In autophagy-inducing rapamycin-treated
NIH3T3 cells, the overexpression of M11 suppressed the
Figure 2. Beclin1 Has a BH3-Like Domain Containing an Atypical Threonine and an Exposed Hydrophobic Patch
(A) A structural comparison of the M11–Beclin1(101–150) (left) and the BCL-X
L
–BAD complexes (right). M11 and BCL-X
L
are shown as surface models.
The Beclin1 and BAD residues shown in sticks correspond to the five BH3 residues that are critical for the interactions with antiapoptotic BCL-2 family
members [21]. They occupy equivalent positions at the BH3-binding groove in the two structures. The surface coloring scheme is as follows: yellow for
Val, Leu, Ile, Tyr, Phe, Trp, Met, and Ala; blue for Lys, Arg, and His; red for Glu and Asp; gray for other amino acids.
(B) Sequence comparison of the BH3-like domain of mouse Beclin1 with various BH3 domains. Conserved residues are highlighted by red or pink
columns. The arrows indicate the five BH3 residues shown in (A). Of these, Thr117 of Beclin1 (red arrow) is not conserved.
(C) Sequence alignment. The BH3-like domains of Beclin1 orthologues are aligned (mm, mouse; hs, human; xl, Xenopus laevis; tr, Takifugu rubripes; dm,
Drosophila melanogaster; sc, Saccharomyces cerevisiae). The arrows at the top indicate the BH3 residues shown in (A). These residues are highly
conserved throughout species, except for Thr117 of mouse Beclin1, which is conserved only in the vertebrates. The conserved hydrophobic residues of
Beclin1 exposed in the structure are indicated by the blue arrows at the bottom.
(D) a-helical wheel representation. The Beclin1 a-helix bound to M11 is compared with the BAD a -helix bound to BCL-X
L
. The Beclin1 helix has a
hydrophobic patch (indicated by an asterisk) on the opposite side of the BH3-binding groove, unlike the BAD helix.
doi:10.1371/journal.ppat.0040025.g002
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Bases for Inhibition of Host Cell Death by M11
Page 5
formation of LC3-II more efficiently than the overexpression
of BCL-2 (Figure 4D). These data collectively demonstrate
that M11 is a more potent autophagy inhibitor compared
with cellular BCL-2, and that the potency directly correlates
with their binding affinity for Beclin1.
Interactions of M11 with BH3 Peptides of Proapoptotic
BCL-2 Relatives
To gain insights into the antiapoptotic activity of M11, we
analyzed the interaction between the apoptosis mediators
BAX and BAK with M11. First, 293T cells were transfected
with HA-tagged BAK or Flag-tagged BAX, together with each
of four different GST-tagged prosurvival BCL-2 proteins
including M11. These proteins, all in the full-length form,
were transiently expressed. A following immunoprecipitation
assay revealed that M11 exhibited a tight interaction with
BAK (Figure 5A, left panel, lane 3) and a comparatively weak
interaction with BAX (Figure 5A, right panel, lane 2). The
M11 binding to BAX and BAK, as expected, depended on its
intact BH3-binding groove, as triple mutations on the groove
abrogated the binding interactions (Figure 5A). Definitely,
the M11 binding to BAK was significantly tighter than the
BCL-2 binding to BAK (Figure 5A, left panel, lane 6).
However, the M11 binding to BAX appeared to be compa-
rable at most or weaker compared with the BCL-2 binding to
BAX (Figure 5A, right panel, lane 5). In this cell-based assay,
KSHV BCL-2 also interacted strongly with BAK (Figure 5A,
left panel, lane 5). However, its interaction with BAX was
barely detected (Figure 5A, right panel, lane 4, and Figure S5),
indicating that KSHV BCL-2 has much poorer affinity for
BAX than M11. These results suggested that M11 could
inhibit BAK strongly but BAX weakly and that the apoptosis
inhibition by KSHV BCL-2 may not be through neutralizing
BAX. Next, we quantified the interactions of M11 with 26-mer
peptides containing the BH3 domain of BAX or BAK. In the
analysis using ITC, M11 interacted with the BAX peptide
weakly, exhibiting a K
D
of 690 nM (Figure 5B). In contrast,
M11 interacted much more tightly with the BAK peptide with
a K
D
of 76 nM (Figure 5B). These measured binding affinities
explain and correlate with the cell-based binding assay using
the full-length proteins of M11, BAX and BAK. We noted that
16-mer peptide (residues 69–84), shorter but spanning the
BH3 domain of BAK, produced a flat titration curve and its
binding affinity for M11 could not be deduced, and thus a
longer BH3-containing sequence of BAK is required for tight
binding to M11. In reflection of the binding assay, the
Figure 3. M11 Interacts Much More Tightly with Beclin1 than BCL-2 and BCL-X
L
(A) ITC analysis. The measurement was carried out by titrating 0.1 mM of M11 or BCL-2 into 5 lM of the indicated Beclin1 fragment. The K
D
values were
deduced from curve fittings of the integrated heat per mol of added ligand.
(B) M11–Beclin1 interface is tighter than that of BCL-X
L
–Beclin1. The structures of M11–Beclin1 fragment (left) and BCL-X
L
–Beclin1 peptide (right) are
compared side by side. In both the structures, the five consensus BH3 residues of the bound a-helices and the side chains of M11 or BCL-X
L
interacting
with those residues are shown as sticks and labeled. Noted are the tighter interactions of the N-terminal region of Beclin1 with the a3 helix of M11 than
the corresponding region in the BCL-X
L
–Beclin1 helix (indicated by dotted circles).
doi:10.1371/journal.ppat.0040025.g003
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Bases for Inhibition of Host Cell Death by M11
Page 6
interaction between M11 and endogenous BAK could be
easily detected in NIH3T3 cells (Figure 5C).
Also using ITC, we next analyzed the interactions between
M11 and the BH3 domain-containing peptides of the eight
well-studied BH3-only proteins BAD, BIK, BIM, BID, BMF,
PUMA, Noxa and Hrk that act upstream of BAX/BAK. These
BH3 peptides, containing 24 to 27 amino acids, are the same
as or 1 to 2 residues longer than those used by Chen et al. for
studying the interactions between the BH3-only proteins and
a cohort of prosurvival BCL-2 proteins [40]. In their study,
the long BH3 peptides did not appear to pose a problem of
reduced helical propensities, because they bound to at least
one of the BCL-2 proteins potently. Given this observation
and the short BH3-binding groove of M11, which can be fully
spanned by 19 residues of Beclin1 (Figure 2A), we conclude
that the length of the BH3 peptides is likely to be optimal. As
shown in Figure 6 and Table S2, M11 interacted with the BIM,
Noxa, BID, BMF and PUMA peptides fairly tightly with the K
D
values ranging from 131–370 nM, while it interacted with the
Hrk peptide rather weakly (K
D
of 719 nM). However, M11 did
not interact or poorly interacted with the BAD and BIK
peptides such that K
D
values could not be deduced. Using an
optical biosensor, Chen et al. previously quantified the
interactions between the entire cohorts of the cellular
antiapoptotic BCL-2 relatives with the BH3 domain peptides
of the BH3-only proteins [40]. A comparison of these data
with our results shows that M11 is dissimilar from any of the
five cellular BCL-2 homologues in the selectivity and affinity
for the BH3 domain peptides (Table S1). For example, while
M11 has high affinity for the Noxa peptide but negligible
Figure 4. M11 Inhibits Autophagosome Formation in NIH3T3 Cells More Efficiently than BCL-2
(A) Light microscopic quantification of autophagy. After transfection with a GFP–LC3 expression plasmid together with the vector encoding the
indicated protein, cells were maintained under normal conditions or treated with 2 lM rapamycin for 4 h. M11(AAA) is an M11 mutant containing three
alanine substitutions at the BH3-binding groove. Autophagy was quantified as the percentage of GFP–LC3 positive cells (top) or as the number of
autophagosomes (GFP–LC3 positive dots) per cell (bottom). The expression of M11 resulted in fewer GFP–LC3 positive cells or spots than the expression
of BCL-2. Data represent mean 6 s.d. of three experiments. Expression levels of M11, M11(AAA) and BCL-2 are shown below.
(B) Confocal microscopic images of the rafamycin treated cells. GFP–LC3 was detected using an inverted fluorescence microscope. Arrows indicate
autophagosomes labeled with GFP–LC3.
(C) Dose response. NIH3T3 cells were transfected with GFP–LC3 expression plasmid together with increasing amount of plasmid encoding the indicated
proteins. At 16–18 h posttransfection, cells were subjected to 2 lM rapamycin treatment for 4 h and autophagy level was quantified as described at (A).
(D) LC3 mobility shift. The whole cells lysates of the rafamycin treated cells were subjected to immunoblotting with anti-LC3 and anti-tubulin antibodies
(left). The ratio of quantified band intensities is also shown (right). The cleaved form (LC3-II) of the LC3 precursor (LC3-I) was undetectable and the level
of LC3-II/LC3-I was far lower in the cells expressing M11 in contrast with the cells expressing BCL-2. Data represent mean 6 s.d. of three experiments. **,
P , 0.005 versus vector (Student t test).
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Bases for Inhibition of Host Cell Death by M11
Page 7
affinity for the BAD peptide, BCL-2 exhibits the opposite
binding affinity for the two peptides (Table S1). Importantly,
M11 binds tightly the BH3 domain peptides of BIM and
PUMA, which have potent cell-killing activity probably owing
to their selectivity for all the five anti-death BCL-2 relatives
[40]. Moreover, M11 exhibited extremely poor binding
affinity for the BH3 domain peptides of BAD and BIK, which
have limited selectivity for BCL-2/BCL-X
L
and relatively poor
apoptotic activity [40].
Discussion
Beclin1 Appears a Main Target of M11
A newly identified function of BCL-2 is the down regulation
of autophagy through their inhibitory binding to Beclin1,
which appears critical for cellular homeostasis [30]. As shown
by others [32] and in this study, the BCL-2/BCL-X
L
interaction
with Beclin1 is quite weak compared with their interactions
with the BH3-only proteins such as BAD and BIM [40]. The
weak interaction explains the recent observation that endog-
enous BH3-only proteins induce autophagy by displacing
Beclin1 from BCL-2/BCL-X
L
[31]. Like the cellular kin, two
viral BCL-2 proteins from cHV68 and KSHV are known to
inhibit autophagy in addition to suppressing apoptotic death
of cells [28,30]. In this study, we provided the structural basis
for the inhibitory interaction of M11 with Beclin1, which is
reminiscent of the canonical interaction between a BH3
peptide and a BH3-binding groove. Significantly, M11 bound
to Beclin1(101–267) more tightly than BCL-2 did. Further-
more, the affinity of binding (K
D
of 40 nM) between M11 and
Beclin1(101–267) was higher than that between M11 and any
of the ten different BH3 peptides used in this study. As a
confirmatory experiment, we carried out a displacement test,
Figure 5. Analyses of the Interactions between M11 or BCL-2 Proteins and BAX/BAK
(A) Cell-based binding assay. 293T cells were transfected with HA-tagged BAK or Flag-tagged BAX together with the indicated GST-tagged prosurvival
BCL-2 proteins. Whole cell lysates were used for immunoprecipitation with anti-GST followed by immunoblotting with anti-HA or anti-Flag. While no
band was detected for the interaction of GST–KSHV BCL-2 with Flag–BAX in this run, a faint band was detected in another run (Figure S5).
(B) ITC analyses of the interactions of M11 with the BH3 peptides of BAX or BAK. The ITC analysis was carried out by titrating 0.1 mM of the indicated
peptides into 5 lM of M11. The ITC run for the titration of the 26-mer BAK peptide is shown. The deduced K
D
values are shown in the table.
(C) M11 interacts with endogenous BAK. NIH3T3 cells were transfected with HA-tagged M11 and whole cell lysates were used for immunoprecipitation
with control rabbit serum or anti-BAK followed by immunoblotting with anti-HA.
doi:10.1371/journal.ppat.0040025.g005
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Bases for Inhibition of Host Cell Death by M11
Page 8
where a complex between two proteins was challenged by
another protein. Consistent with our affinity measurement,
the M11–Beclin1(101–267) complex remained intact when it
was incubated with the BIM, BID or Noxa peptide (Figure
S6A). In contrast, the BCL-2–Beclin1(101–267) or BCL-X
L
Beclin1(101–267) complex was easily disrupted by BAD or
BIM peptide (Figure S6B). Conceivably, M11 could negate the
proautophagic role of the BH3-only proteins under apopto-
sis-inducing conditions in contrast with BCL-2/BCL-X
L
. The
observed robust interaction of M11 with the Beclin1 fragment,
which correlates with its strong antiautophagic effect in
NIH3T3 cells (Figure 4), suggests that Beclin1 may be a main
target of M11 and that the inhibition of autophagy may
contribute to the viral infection of cells.
Viral BCL-2 homologues, including M11, share limited
sequence homology with the cellular kin [2]. Nonetheless, two
available structures of KSHV BCL-2 and M11 have demon-
strated that they are structurally homologous to the cellular
kin and possess a prominent surface groove which binds the
BH3 doma in peptides from proapoptotic BCL-2 family
members [7,41]. While the known BCL-2 homologues
encoded by alpha and gamma herpesviruses exhibit only
20–30% overall sequence homology with each other [2], we
noted that the residues of M11 significantly involved in the
interactions with the Beclin1 fragment share 60–90%
sequence similarity with the corresponding residues of the
other herpesviral BCL-2 proteins (Figure S7). This observa-
tion raises a possibility that at least some alpha and gamma
herpesviral BCL-2 homologues could interact with the BH3-
like domain of Beclin1. In addition, some structural viral
mimics of BCL-2, such as M11L of Myxoma virus [33] and N1
of Vaccinia virus [42], might also interact with Beclin1
through their BH3-binding groove.
M11 Broadly Engages Proapoptotic BCL-2 Proteins
The underlying mechanism of how viral BCL-2 homologues
or mimics suppress apoptosis is not well understood. Perhaps
M11L of Myxoma virus is best characterized in this regard.
Through structural and bioche mical analyses, M11L was
shown to bind BAX, BAK and BIM proteins or peptides
tightly but not the other proapoptotic BH3-only proteins
[33]. Using a panel of M11L mutants containing an amino
acid substitution at the BH3-binding groove, it was demon-
strated that the prosurvival action of M11L largely depended
on binding BAX and BAK [33]. The observation is consistent
with a general expectation that viral BCL-2 would prefer to
target BAX/BAK rather than the upstream BH3-only proteins
[1]. In contrast with the binding selectivity of M11L, our
quantitative binding analysis indicated that M11 primarily
targets BAK, but not BAX, and broadly engages the BH3-only
proteins except for BAD and BIK (Figure 7). How could M11,
having the weak binding affinity for BAX, antagonize
Figure 7. Model for M11 Action Mechanism
The binding analyses presented in this study suggest that M11 antagonizes cell death by simultaneous inhibition of apoptosis and autophagy. The
varied thickness of the arrows denoting the negative regulation by M11 indicates the inhibitory potency according to the K
D
values determined in this
study and shown next to the arrows. Dashed line is used to indicate that BAX may be inhibited by M11 but only weakly. Although not indicated in the
figure, cellular BCL-2 proteins, protected by M11, may sequester and inhibit BAX (see text). PI(3)KCIII and UVRAG stand for class III phosphatidylinositol
3-kinase and UV irradiation resistance-associated gene, respectively.
doi:10.1371/journal.ppat.0040025.g007
Figure 6. ITC Analyses of the Interactions of M11 with the BH3 Peptides of BH3-Only Proteins
Representative ITC runs for the interaction of M11 with the Noxa and BAD peptides are shown, and the K
D
values determined by this method are
summarized in the table.
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Bases for Inhibition of Host Cell Death by M11
Page 9
apoptosis of cells following the rise of the concentration of
the activated BH3-only proteins under apoptosis-inducing
conditions? We speculate that the neutralization of a subset
of the BH3-only proteins (including BIM, BID, BMF, PUMA
and Noxa) by M11 should prevent them from engaging their
cellular prosurvival BCL-2 targets, and this protection would
allow some fractions of the prosurvival proteins to keep
suppressing the activation of BAX. This possibility is relevant
to the suggestion that all the BCL-2 relatives keep BAX in
check, whereas only BCL-X
L
and MCL-1 inhibit BAK
according to the so-called indirect activation model [18]. In
this scenario, although M11 cannot neutralize BAD and BIK,
MCL-1, having very low affinity for BAD and BIK [40], and
other prosurvival protein molecules saved by M11 can inhibit
BAX when M11 is expressed in the infected cell. An
alternative possibility is that M11 inhibits the BAX activation
by neutralizing BIM, BID, and PUMA, which are believed to
directly activate BAX/BAK according to the hierarchical
regulatory scheme [17]. Although further investigations may
shed light on this important issue, the data presented here,
including the weak interaction of KSHV BCL-2 with BAX
(Figures 5A and S5), suggest that viral BCL-2 homologues may
not necessarily target both BAX and BAK to suppress
apoptosis.
Concluding Remarks
We provided structural and biochemical bases for how M11
may subvert the antiviral host defense mechanisms, which is
likely to involve both apoptosis and the Beclin1-dependent
autophagy. Further studies are ne cessary to assess t he
importance of the Beclin1-dependent autophagy as an
antiviral measure and to understand the consequences of the
robust interaction of M11 with Beclin1 in the establishment
and/or maintenance of the viral chronic life cycle. Our work
provides a rational ground for future investigation to learn
whether the inhibition of the Beclin1-dependent autophagy is
the unique property of M11 and KSHV BCL-2 or is a general
feature of other viral BCL-2 homologues or mimics.
Materials and Methods
Preparation, crystallization, and structure determination of the
M11–Beclin1(101–150) complex. The DNA fragments coding for M11
(residues 1–137) and mouse Beclin1 (residues 101–150) were cloned
into pET30a (Novagen) and pPROEX HTa (Invitrogen), respectively.
From these vectors, a two-promoter vector was constructed for
coexpression of the two proteins. The protein complex was produced
in E. coli BL21(DE3) strain (Novagen) at 21 8C overnight and purified
using a Ni-NTA column (QIAGEN), a Hitrap Q anion exchange
column (Amersham Pharmacia) and a Mono Q anion exchange
column (Amersham Pharmacia), equilibrated with 20mM Tris-HCl
(pH 8.0), 220mM NaCl and 1mM dithiothreitol. Crystals of the
complex were obtained by the hanging-drop vapor diffusion method
at 24 8C by mixing and equilibrating 1 ll of each of the protein
solution (10 mg/ml) and a precipitant solution containing 25% (w/v)
polyethylene glycol 3350, 0.2 M magnesium chloride, and 0.1 M
imidazole (pH 7.0). Before data collection, the crystals were immersed
briefly in a cryoprotectant solution, which was the reservoir solution
plus 10% glycerol. A diffraction data set at 2.3 A
˚
resolution was
collected on the beamline 4A at the Pohang Accelerator Laboratory,
Korea, and processed using the programs DENZO and SCALEPACK
[43]. The structure was determined by the molecular replacement
method with the CCP4 version of MolRep [44] using the structure of
M11 [7] as a search model. Subsequently, model building and
refinement were carried out using the programs O [45] and CNS
[46]. The final model does not include residues 1–4 and 136–137 of
M11, and residues 101–105 and 125–150 of Beclin1, whose electron
densities were not observed or were very weak.
Preparation, crystallization, and structure determination of the
BCL-X
L
–BAD complex. The DNA fragment coding for mouse BCL-X
L
(residues 1–196) was cloned into pPROEX HTa. This construct was
used as a template for deletion mutagenesis to produce BCL-X
L
lacking the internal long loop (residues 45–84) and the C-terminal tail
region (residues 197–235). DNA fragment coding for mouse BAD
(residues 43–204; corresponding to residues 1–168 of human BAD)
was cloned into pET30a. A two-promoter vector was constructed
from these two vectors. The protein complex was expressed in the E.
coli BL21(DE3) RIG strain (Novagen) at 21 8C overnight and purified
using a Ni-NTA column, a Hitrap Q anion exchange column and a
HiLoad26/60Superdex75gelltrationcolumn(Amersham
Pharmacia), equilibrated with 20mM Tris-HCl (pH 8.0), 100mM NaCl,
and 1mM dithiothreitol. Crystals of the complex were obtained by the
hanging-drop vapor diffusion met hod at 4 8Cbymixingand
equilibrating 1 ll of each of the protein solution (5 mg/ml) and a
precipitant solution containing 10% (w/v) polyethylene glycol 1000
and 10% (w/v) polyethylene glycol 8000. Before data collection, the
crystals were immersed briefly in a cryoprotectant solution, which
was the reservoir solution plus 16% glycerol. A diffraction data set at
2.3 A
˚
was collected on the beamline 41XU at the Spring-8, Japan. The
structure was determined by the molecular replacement using the
structure of BCL-X
L
[47] as a search model. The final model does not
include residues 31–44 of BCL-X
L
, and residues 43–136 and 164–204
of BAD. Crystallographic data statistics are summarized in Table 1.
Purification of BCL-2 family proteins and Beclin1 fragment. Each
of the DNA fragments coding for M11 (residues 1–137), mouse BCL-
X
L
(residues 1–44 and 85–196) or mouse Beclin1 (residues 101–267)
was cloned into pPROEX HTa. A plasmid containing the DNA
segment coding for human BCL-2 (residues 1–50 and 92–207) was also
constructed. The resulting protein lacks the internal long loop
(residues 51–91) and contains a replacement of residues 35–50 with
residues 33–48 of BCL-X
L
, which was necessary for the solubility of
the protein as reported earlier [48]. Each construct was introduced
into the E. coli BL21(DE3) strain. The proteins were expressed at 21 8C
overnight and purified using a Ni-NTA column and a Hitrap Q anion
exchange column.
Peptides. Synthetic peptides of 25-mer (residues 101–125 of
Beclin1), 20-mer (residues 106–125 of Beclin1), 16-mer (residues
69–84 of BAK), 26-mer (residues 65–90 of BAK), 26-mer (residues 52–
77 of BAX), 27-mer (residues 137–163 of BAD), 25-mer (residues 45–
69 of BIK), 26-mer (residues 139–164 of BIM), 25-mer (residues 80–
104 of BID), 24-mer (residues 214–237 of BMF), 26-mer (residues 130–
155 of PUMA), 26-mer (residues 16–41 of Noxa), and 26-mer (residues
26–51 of Hrk) were purchased from Peptron (Korea).
Isothermal titration calorimetry. All measurements were carried
out at 25 8C on a MicroCalorimetry System (MicroCal). Protein
samples were dialyzed against the solution containing 20 mM Tris-
HCl (pH 7.4) and 100 mM NaCl. The samples were degassed for 20
min and centrifuged to remove any residuals prior to the measure-
ments. Dilution enthalpies were measured in separate experiments
(titrant into buffer) and subtracted from the enthalpies of the
binding between the protein and the titrant. Data were analyzed
using the Origin software (OriginLab Corp.).
Autophagy analyses. Autophagy was assessed by GFP–LC3 redis-
tribution and LC3 mobility shift. For GFP–LC3 redistribution assay,
NIH3T3 cells were transfected with a GFP–LC3 expression plasmid
together with the vector encoding BCL-2, M11, or M11(AAA). At 16–
18 h posttransfection, GFP–LC3 in the cells grown under normal and
2 lM rapamycin-treated medium containing 1% FBS for 4 h was
detected using an inverted fluorescence microscope. The percentage
of GFP–LC3-positive cells with punctuate staining was determined in
three independe nt experiments. To quantify GFP–LC3-positive
autophagosomes per transfected cell, six random fields representing
200 cells were counted. For the LC3 mobility shift assay, NIH3T3 cells
transfected with the vector encoding BCL-2, M11 or M11(AAA) were
treated for 30 min on ice, lysed with 1% Triton X-100 and then
subjected to immunoblot analysis with an antibody against LC3
(Santa Cruz Biotech).
Immunoprecipitation assay. Each of fusion protein GST–BCL-2,
GST–KSHV BCL-2, GST–M11 and GST–M11(AAA) was cloned into
pcDNA5/FRT/TO (Invi trogen) and overexpressed in 293T cells
together with HA-tagged BAK or Flag-tagged BAX. HA–M11, HA–
M11(AAA) and HA–BCL-2 proteins were also cloned into pcDNA5/
FRT/TO and overexpressed in NIH3T3 cells, respectively. Cells were
harvested and lysed in NP40 buffer supplemented with a complete
protease inhibitor cocktail (Roche). Immunodetection was achieved
with anti-Flag (1:5000) (Sigma), anti-HA (1:5000), anti-GST (1:2000),
anti-tubulin (1:1000), anti-BAK (1:100), or anti-Beclin1 (1:500) (Santa
Cruz Biotech), which was incubated at 40 8C for 8–12 h. The proteins
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Bases for Inhibition of Host Cell Death by M11
Page 10
were visualized by a chemiluminescence reagent ( Pierce) and
detected by LAS 3000 (Fujifilm).
Circular dichroism spectroscopy. Data were collected on a JASCO
model J-810 spectropolarimeter with a 0.2 cm cuvette. CD spectrum
was recorded over the range of 200–250 nm in a nitrogen atmosphere
with peptides dissolved in 40 mM sodium phosphate buffer (pH 7.0)
containing 30% TFE at the concentration of 0.1 mg/mL. The
spectrum was the accumulation of three scans corrected by
subtracting signals from the buffer control. The law CD signal at
222 nm (in millidegrees) was converted to mean residue ellipticity
([h]
obs
, in deg
.
cm
2 .
dmol
1
) using the equation
½h
obs
¼ 100ðsignal at 222 nmÞ=Cnl ð1Þ
where C is the peptide concentration (in millimolarity), n is the
number of residues in the peptide, and l is the pathlength (in cm).
The contents of helix (F
helix
) was calculated using the equation
F
helix
¼ð½h
obs
½h
coil
Þ=ð½h
helix
½h
coil
Þð2Þ
where [h]
helix
represents the mean residue ellipticity for a complete
helix of infinite length at 0 8C(42,500(13/n) deg
.
cm
2 .
dmol
1
) and
[h]
coil
is the ellipticity of a complete random coil at 0 8C (640 deg
.
cm
2
.
dmol
1
) [49,50].
Supporting Information
Figure S1. Helicity of Beclin1 Peptides
The contents of a-helix of the indicated Beclin1 peptides in 30% TFE
were calculated using the mean residue ellipticity at 222 nm acquired
from the displayed circular dichroism spectra. The Beclin1(101–125)
peptide has considerably higher helical contents than the Be-
clin1(106–125) peptide.
Found at doi:10.1371/journal.ppat.0040025.sg001 (257 KB TIF).
Figure S2. Interactions of BCL-X
L
/MCL-1 with BAK or BIM BH3
Domain
BCL-X
L
and MCL-1 are shown as surface models. The BAK and BIM
residues shown in sticks are the five conserved residues in the BH3
domains. They occupy equivalent positions at the BH3-binding
grooves as those in BAD (Figure 2A). The surface coloring scheme is
the same as that of Figure 2A.
Found at doi:10.1371/journal.ppat.0040025.sg002 (4.4 MB TIF).
Figure S3. The Beclin1 a-Helix Bound to M11 Has a Hydrophobic
Patch
M11 and BCL-X
L
are shown as surface models. The Beclin1 and BAD
residues are shown as sticks and labeled. Three exposed hydrophobic
residues of Beclin1 forming a hydrophobic patch on the opposite side
of the BH3-binding groove are indicated by red ellipses and labels. In
contrast, the bound BAD helix does not contain any exposed
hydrophobic residue.
Found at doi:10.1371/journal.ppat.0040025.sg003 (3.6 MB TIF).
Figure S4. Interactions of the Beclin1 Helix (Shown in Green) with
M11 (Left) or BCL-X
L
(Right)
The two views are shown in the similar orientation along with the
final 2F
o
-F
c
map (1 r) at 2.3 A
˚
and 2.5 A
˚
, respectively. A portion of the
Beclin1-binding interface involving the a3 helix (dotted circles) of
BCL-X
L
exhibits poor electron density in contrast with the
corresponding region of M11.
Found at doi:10.1371/journal.ppat.0040025.sg004 (3.6 MB TIF).
Figure S5. KSHV BCL-2 Interacts with BAX Much Less Tightly than
M11
293T cells were transfected with Flag-tagged BAX together with the
GST–M11 or GST–KSHV BCL-2. Whole cell lysates were used for
immunoprecipitation with anti-GST followed by immunoblotting
with anti-Flag.
Found at doi:10.1371/journal.ppat.0040025.sg005 (215 KB TIF).
Figure S6. Displacement Test
(A) Three BH3 peptides failed to displace Beclin1(101–267) bound to
M11. After M11 was mixed with Beclin1(101–267) at 1:1 molar ratio
and incubated for 1 h, the BIM, BID or Noxa BH3 peptide was added
to the mixture and incubated for an additional 1 h. The M11–
Beclin1(101–267) complex remained intact.
(B) BIM and BID peptides displaced Beclin1(101–267) bound to BCL-
2 or BCL-X
L
. After BCL-2 and BCL-X
L
were mixed with Beclin1(101–
267) at 1:4 molar ratio and incubated for 1 h, the BAD or BIM BH3
peptide was added to the mixtures. The complexes between each of
the BH3 peptides and BCL-2 or BCL-X
L
appeared. The numbers
above the gels indicate the final concentrations of the indicated
proteins and peptides. The schematic drawings are shown to help to
understand the experimental scheme and results.
Found at doi:10.1371/journal.ppat.0040025.sg006 (1.8 MB TIF).
Figure S7. Multiple Sequence Alignment
The sequences of eight alpha and gamma herpesviral BCL-2
homologues are aligned (M11; KSHV BCL-2; RRV ORF16, rhesus
rhadinovirus open reading frame 16; BHV4 BORFB2, bovine
herpesvirus 4 BORFB2; HVS ORF16, saimiriine herpesvirus ORF16;
MeHV BCL-2, meleagrid herpesvirus BCL-2; EBV BHRF1, Epstein-
Barr virus BHRF1; EHV2 ORFE4, equid herpesvirus 2 ORF E4). The
asterisk marks indicate the 11 residues of M11 that make hydrophobic
contacts with the Beclin1 helix within 4.0 A
˚
or hydrophilic contacts
with Asp119 of Beclin1. The pink bars indicate the alpha helices in the
M11 structure and the black bar indicates hydrophobic tails (HT). The
residues exhibiting more than 70% of similarity are shaded.
Found at doi:10.1371/journal.ppat.0040025.sg007 (586 KB TIF).
Table S1. List of the Binding Affinities for the Interactions between
the BH3 Peptides and M11 or Cellular BCL-2 Family Proteins
The binding affinities for M11 are absolute values in K
D
determined
by ITC, while those for the other cellular BCL-2 proteins are relative
values determined by competitive binding assays reported by Chen et
al. [40]. Therefore, the binding profiles, not the binding affinities,
have to be compared.
Found at doi:10.1371/journal.ppat.0040025.st001 (125 KB DOC).
Table S2. List of Thermodynamics Values Determined by ITC for the
Interactions between M11 or BCL-2 and the Indicated Fragment or
Peptides
Found at doi:10.1371/journal.ppat.0040025.st002 (211 KB DOC).
Accession Numbers
The coordinates of the M11–Beclin1 fragment structure and the BCL-
X
L
–BAD structure have been deposited in the Protein Data Bank
(http://www.rcsb.org/pdb/) with the accession codes 3BL2 and 2BZW,
respectively. The accession numbers for the coordinates for the
structures mentioned in this article are M11 (2ABO), BCL-X
L
(1AF3),
BCL-X
L
–Beclin1 (2P1L), BCL-X
L
–BAK (1BXL), BCL-X
L
–BIM (1PQ1),
MCL-1–BIM (2NL9), and KSHV BCL-2 (1K3K). The National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) acces-
sion numbers for the protein sequences in the sequence databases are
mouse Beclin1 (NP_062530), BCL-X
L
(NP_033873), BAX
(NP_031553), BAK (NP_031549), BAD (NP_031548), B IK
(NP_031572), BIM (NP_997563), BID (NP_031570), BMF
(NP_612186), PUMA (NP_573497), Noxa (NP_067426), Hrk
(NP_031571), human Beclin1 (NP_003757), BCL-X
L
(NP_612815),
BCL-2 (NP_000624), MCL-1 (NP_068779), BAK (NP_001179), BIM
(NP_619527), xlBeclin1 (AAH73292), trBeclin1 (NP_001032963),
dmBeclin1 (NP_651209), scBeclin1 (BAA32104), M11(AAF19336),
KSHV BCL-2 (NP_572068), RRV ORF16 (AAF59994 ), BHV4
BORFB2 (NP_076508), HVS ORF16 (CAA73630), MeHV BCL-2
(NP_073365), EBV BHRF1 (CAD53396), and EHV2 ORFE4
(NP_042601).
Acknowledgments
This study made use of beamline 4A at the Pohang Accelerator
Laboratory in Korea.
Author contributions. BK, JSW, JUJ, and BHO conceived and
designed the experiments. BK, JSW, CL, KHL, HSH, XE, and KSK
performed the experiments. BK, JSW, JUJ, and BHO analyzed the
data. BK, JSW, and BHO wrote the paper.
Funding. This work was supported by Creative Research Initiatives
(Center for Biomolecular Recognition) of MOST/KOSEF of Korea (to
BHO) and by Functional Proteomics Center (The 21 century Frontier
Research Program) from MOST of Korea (to KSK). BK, JSW, and KHL
were supported by the Brain Korea 21 Project.
Competing interests. The authors have declared that no competing
interests exist.
PLoS Pathogens | www.plospathogens.org February 2008 | Volume 4 | Issue 2 | e250011
Bases for Inhibition of Host Cell Death by M11
Page 11
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PLoS Pathogens | www.plospathogens.org February 2008 | Volume 4 | Issue 2 | e250012
Bases for Inhibition of Host Cell Death by M11
Page 12
    • "Overexpression of antiapoptotic Bcl-2 family proteins has been demonstrated to contribute to prolonged cell survival, resistance to apoptosis, cancer development and progression, and decreased sensitivity to chemotherapeutics in cancer cells [11] [12]. The proapoptotic BAD protein interact with the hydrophobic binding groove of Bcl homologs (Bcl-2 and Bcl-xL) [13] and disrupts the integrity of mitochondria to initiate the release of cytochrome C into the cytoplasm to induce apoptosis [14] [15]. Therefore, targeting Bcl- 2/Bcl-xL proteins using small molecules that can interact with their hydrophobic socket may serve as agents with therapeutic potential to induce apoptosis in cancer cells. "
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    No preview · Article · Dec 2015 · Bioorganic & Medicinal Chemistry Letters
  • Source
    • "ICP34.5 binding to Beclin 1 inhibits the formation of autophagosomes in neurons, suggesting that the virus has evolved to actively inhibit autophagy. Other viral proteins inhibiting through Beclin 1 binding include Bcl-2 homologs, such as the KSHV orf16 protein and the MHV-68 M11 protein (Ku et al., 2008; Su et al., 2014). In addition to ICP34.5's "
    [Show abstract] [Hide abstract] ABSTRACT: Studies of the cellular autophagy pathway have exploded over the past twenty years. Now appreciated as a constitutive degradative mechanism that promotes cellular homeostasis, autophagy is also required for a variety of developmental processes, cellular stress responses, and immune pathways. Autophagy certainly acts as both an anti-viral and pro-viral pathway, and the roles of autophagy depend on the virus, the cell type, and the cellular environment. The goal of this review is to summarize, in brief, what we know so far about the relationship between autophagy and viruses, particularly for those who are not familiar with the field. With a massive amount of relevant published data, it is simply not possible to be comprehensive, or to provide a complete "parade of viruses", and apologies are offered to researchers whose work is not described herein. Rather, this review is organized around general themes regarding the relationship between autophagy and animal viruses. Copyright © 2015. Published by Elsevier Inc.
    Preview · Article · Apr 2015 · Virology
  • Source
    • "However, the utilization of autophagic membranes has not been reported so far for DNA viruses. Indeed, herpes viruses rather seem to encode Bcl-2 homologues that inhibit autophagosome formation via their binding to Atg6/Beclin-1 (E et al., 2009; Ku et al., 2008; Orvedahl et al., 2007; Pattingre et al., 2005 "
    [Show abstract] [Hide abstract] ABSTRACT: Epstein Barr virus (EBV) persists as a latent herpes virus infection in the majority of the adult human population. The virus can reactivate from this latent infection into lytic replication for virus particle production. Here, we report that autophagic membranes, which engulf cytoplasmic constituents during macroautophagy and transport them to lysosomal degradation, are stabilized by lytic EBV replication in infected epithelial and B cells. Inhibition of autophagic membrane formation compromises infectious particle production and leads to the accumulation of viral DNA in the cytosol. Vice versa, pharmacological stimulation of autophagic membrane formation enhances infectious virus production. Atg8/LC3, an essential macroautophagy protein and substrate anchor on autophagic membranes, was found in virus preparations, suggesting that EBV recruits Atg8/LC3 coupled membranes to its envelope in the cytosol. Our data indicate that EBV subverts macroautophagy and uses autophagic membranes for efficient envelope acquisition during lytic infection.
    Full-text · Article · Dec 2014 · EBioMedicine
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