Cellular Signalling 24 (2012) 14201425

Contents lists available at SciVerse ScienceDirect

Cellular Signalling
journal homepage: www.elsevier.com/locate/cellsig

Review

A molecular view on signal transduction by the apoptosome

Thomas F. Reubold, Susanne Eschenburg
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany

a r t i c l e

i n f o

Article history:
Received 7 February 2012
Accepted 5 March 2012
Available online 13 March 2012
Keywords:
Apoptosis
Apaf-1
Apoptosome
Caspase activation

a b s t r a c t
Apoptosomes are signaling platforms that initiate the dismantling of a cell during apoptosis. In mammals,
assembly of the apoptosome is the pivotal point in the mitochondrial pathway of apoptosis, and is prompted
by binding of cytochrome c to the apoptotic protease-activating factor 1 (Apaf-1) in the presence of ATP. The
resulting wheel-like heptamer of seven molecules Apaf-1 and seven molecules cytochrome c binds and activates the initiator caspase-9, which in turn ignites the downstream caspase cascade. In this review we discuss
the molecular determinants for the formation of the mammalian apoptosome and caspase activation and
describe the related signaling platforms in ies and nematodes.
2012 Elsevier Inc. All rights reserved.

Contents
1.
Introduction . . . . . . . . . . . . . . . .
2.
Apaf-1 . . . . . . . . . . . . . . . . . . .
3.
Cytochrome c . . . . . . . . . . . . . . .
4.
ATP . . . . . . . . . . . . . . . . . . . .
5.
Structure of the Apaf-1 apoptosome . . . . .
6.
Caspase activation by the Apaf-1 apoptosome
7.
The Apaf-1 orthologs Dark and Ced-4 . . . .
8.
Concluding remarks . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . .

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1. Introduction
Apoptosis is a highly regulated cellular mechanism in metazoans
used to terminate superuous or unwanted cells in a controlled way
[1]. Apoptosis is crucial for normal human embryonic development
[2,3] and the development of the immune system [4]. Deregulation
of apoptosis is associated with severe pathologic conditions, e.g. cancer and neurodegenerative diseases [58]. Two different pathways
have emerged, which both lead to the activation of members of a
class of cystein-dependent aspartate-specic proteases (caspases).
Activation of the caspase cascade ultimately leads to the orderly degradation of the cell [9].
The extrinsic apoptotic pathway is induced by binding of death
ligands to the ectodomains of their cognate death receptors [10]
and leads to the activation of the initiator caspases 8 and 10.

Corresponding author. Tel.: + 49 511 532 8655; fax: + 49 511 532 2909.
E-mail address: eschenburg.susanne@mh-hannover.de (S. Eschenburg).
0898-6568/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellsig.2012.03.007

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The intrinsic or mitochondrial apoptotic pathway is triggered by

different kinds of cellular stress e.g. DNA damaging agents
including UV irradiation and cytotoxic drugs (reviewed in [11]).
Predominance of pro-apoptotic stimuli eventually lead to the Bax
(Bcl-2-associated x protein)- or Bak (Bcl-2 antagonist killer)-induced
permeabilization of the outer mitochondrial membrane [12,13]. Permeabilization is followed by the release of cytochrome c from the
intermembrane space into the cytosol [13]. Binding of cytochrome c
to cytosolic Apaf-1 (apoptotic protease-activating factor 1) in the
presence of ATP yields a large complex of 1 MDa termed apoptosome,
which serves as activation platform for procaspase-9 [14]. The holoapoptosome formed by the apoptosome and caspase-9 in turn activates the effector caspase-3 [15].
In this review we summarize the current knowledge about the
constituents of the mammalian holo-apoptosome and discuss their
role in the transduction of the death signal in the mitochondrial pathway of apoptosis. In doing so we highlight recent structural data and
their implications for the mechanism of apoptosome formation and
subsequent caspase activation.

T.F. Reubold, S. Eschenburg / Cellular Signalling 24 (2012) 14201425

2. Apaf-1
Upon discovery of the executioner caspase-3 the molecular determinants for its activation were still obscure [16,17]. The molecules,
which eventually activate caspase-3 in the mitochondrial branch of
apoptosis, were subsequently identied as procaspase-9 [18], cytochrome c [19], and Apaf-1 [20]. Apaf-1 was recognized as human
counterpart of Ced-4 of the nematode Caenorhabditis elegans [21].
Based on sequence homology Apaf-1 has been grouped into the
STAND (signal transduction ATPases with numerous domains) family
of proteins [22]. Whether the STAND family constitutes a subfamily of
AAA+ (ATPases associated with diverse cellular activities) ATPases is
debated [23,24]. Apaf-1 is expressed in various tissues and exists in at
least four different splice forms [25]. The residue numbering throughout this review refers to the longest isoform termed Apaf-1-XL (residues 11248). Apaf-1 contains three main domains: the N-terminal
caspase recruitment domain (CARD), the nucleotide binding and oligomerization domain (NOD), and the C-terminal regulatory WD40 repeat domain (WRD) (Fig. 1). The CARD belongs to the death domain
superfamily whose members share a common basic fold consisting
of six bundled -helices [26]. The Apaf-1 CARD binds its counterpart
in procaspase-9 via homotypic interactions mostly involving charged
residues [27]. The NOD (residues 108586) harbors a binding site for
adenine nucleotides and mediates oligomerization of Apaf-1 into the
apoptosome complex.
Several years ago the crystal structure of an N-terminal fragment
of human Apaf-1 comprising the CARD and the NOD was determined
[28]. The structure revealed a compact fold of the NOD and that the
NOD can be divided into four subdomains. These subdomains were
termed nucleotide-binding domain (NBD, residues 108284), helical
domain 1 (HD1, residues 285365), winged-helix domain (WHD, residues 366450), and helical domain 2 (HD2, residues 451586). The
nucleotide binding site of the NOD was occupied with an ADP molecule, which was in contact with residues from all four subdomains.
Since the entire regulatory WRD was missing, the explanation of
how Apaf-1 inhibits itself until cytochrome c binds had to wait until
a crystal structure of full-length Apaf-1 was solved.
The crystal structure of full-length murine Apaf-1 in the absence of
cytochrome c and exogenous nucleotide [29] recently showed that
the tandem -propellers of the WRD do not block the CARD as presumed previously. Rather, the WRD acts as a clamp which holds the
subdomains of the NOD in place. Thus, the WRD prevents the conformational rearrangement of the NOD which is required for apoptosome formation [30,31] (see below for further details).
3. Cytochrome c
The identication of cytochrome c as a crucial component of the
mitochondrial pathway of apoptosis came as a surprise [19]. Given
the well known function of cytochrome c in the respiratory chain, a
role as signaling molecule appeared remarkable. However, the fact
that cytochrome c is, in a healthy cell, conned to the mitochondrial
intermembrane space while the executing part of the apoptosis machinery is located in the cytosol makes the molecule well suited as decisive start signal. The strict spatial separation of signal and
machinery minimizes the chance for accidental activation of the
death cascade.
Apaf-1 is able to bind cytochrome c both in the presence and absence of nucleotide [20,32,33]. It was soon recognized that cytochrome c binds to the WRD and that this interaction is required to
relieve the autoinhibition of Apaf-1 imposed by its WRD, since deletion of the WRD abolishes the need for cytochrome c in the assembly
process [34,35]. Biophysical studies assessing the binding characteristics of cytochrome c to Apaf-1 suggested a stoichiometry of two cytochrome c molecules per molecule Apaf-1 [33,36]. However,
subsequent cryo-EM studies clearly showed a 1:1 stoichiometry

1421

without any indication for a secondary cytochrome c binding site on

Apaf-1 [30,31]. Globular density, which may be attributed to cytochrome c, is visible only between the -propellers of individual
Apaf-1 molecules within the apoptosome (see reference [31]). This
observation is supported by the crystal structure of full length Apaf1 [29]. The faces of the -propellers WD1 and WD2 show pronounced
negative charges as would be expected for efcient binding of the
positively charged cytochrome c.
Binding of cytochrome c appears to cause a rotation of -propeller
WD1 [29], which detaches the NBDHD1 subunit of the NOD from the
propeller clamp (Fig. 1). The rotation of -propeller WD1 enables and
even actuates subsequent rotation of the NBDHD1 subunit, which
eventually leads to the extended conformation of Apaf-1 seen in the
apoptosome [29,31]. Furthermore, cytochrome c might prevent reclosing of the opened NOD, since the rotated position of WD1 is sterically incompatible with the closed conformation of the NOD.

4. ATP
A second crucial molecular event during apoptosome formation is
the exchange of (d)ADP bound to Apaf-1 for exogenous (d)ATP [37].
The origin of the bound diphosphate is still a subject of controversy.
According to a study performed in the lab of Xiadong Wang, monomeric Apaf-1 is loaded with dATP [38]. Here, binding of cytochrome
c prompts Apaf-1 to perform a single round of nucleotide hydrolysis.
The hydrolytic energy would be needed to prime Apaf-1 for the ensuing large conformational changes, which in turn are then triggered by
replacing dADP for exogenous dATP. Several other studies contradict
these ndings showing that recombinant monomeric full-length
Apaf-1 as well as WD40-deleted Apaf-1 puried from different insect
cell strains and from Escherichia coli contain ADP [28,29,39,40]. Consistently, a low but steady (d)ATPase activity of monomeric Apaf-1
in the absence of cytochrome c has been described [4043].
For apoptosome assembly the bound ADP has to be exchanged for
an exogenous nucleoside triphosphate in the presence of cytochrome
c. Not only ATP or dATP but also non-hydrolyzable ATP analogs like
AppNHp or AppCp fulll this function, which emphasizes that chemical energy derived from nucleotide hydrolysis is not required at all
during the assembly process. Rather, the presence of a -phosphate
alone is sufcient to allow apoptosome formation [40].
The molecular effect of the nucleotide exchange is still obscure.
However, comparing the nucleotide binding sites of different AAA+
ATPases and STAND proteins, the notion emerges that the determining factor is a polar interaction of the -phosphate with a sensor residue. This residue is located at the tip of -strand 4 as part of the socalled sensor 1 motif. In AAA+ ATPases the sensor residue is mostly
an asparagine, but serine, threonine, aspartate, or histidine are also
found [44,45]. Several studies suggest that sensor 1 is directly involved in ATP hydrolysis, since its mutation strongly decreases cooperative ATPase activity in different proteins [4648]. Most members
of the AAA+ ATPase related family of STAND ATPases, to which
Apaf-1 belongs, possess an arginine as sensor residue [49,50]. In
Apaf-1 this arginine (R265) would be in interaction distance to the
-phosphate group of an ATP molecule in the nucleotide binding
site [29]. Such an interaction is also visible within crystal structures
of Ced-4, both in the Egl-1 inhibited dimer [51] and in the apoptosome [52], as well as in the EM-derived model of the human apoptosome [31].
The importance of the sensor arginine has been conrmed for
Apaf-1 and other STAND proteins. Functional analysis of an Apaf-1
R265S mutant revealed that the mutant protein does not yield functional apoptosomes [29]. In the bacterial transcription regulator
MalT mutation of the putative sensor arginine R160 to alanine
resulted in loss of activation [53]. The same was observed for the corresponding mutation (R313A) in the plant resistance protein I-2 [54].

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T.F. Reubold, S. Eschenburg / Cellular Signalling 24 (2012) 14201425

T.F. Reubold, S. Eschenburg / Cellular Signalling 24 (2012) 14201425

Different to the sensor in AAA+ ATPases, the sensor arginine of

Apaf-1 and of the related Ced-4 does not seem to have a hydrolytic
function. Apaf-1 has no hydrolytic activity in the assembled state
and also Apaf-1 assembly does not rely on nucleotide hydrolysis
[39,40]. Ced-4, the only STAND protein for which atomic structures
are available for both the inhibited and the assembled state, does
not seem to possess hydrolytic activity at all, since in both states the
bound nucleotide is ATP [51,52]. These circumstances point to a stabilizing role of the sensor arginine rather than a contribution to any catalytic activity.
5. Structure of the Apaf-1 apoptosome
The rst evidence for the existence of a high molecular weight
complex as the caspase-activating species of Apaf-1 came from in
vitro studies using recombinant puried proteins [43]. Gel ltration
of Apaf-1 showed that the apparent molecular weight shifted from
that of monomeric Apaf-1 to about 1 MDa if cytochrome c and dATP
were added. The availability of in vitro reconstituted apoptosome enabled the group of Christopher Akey to generate a rst cryo-EM map to
27 that revealed that the apoptosome resembles a wheel with seven
spokes. Each of the spokes consists of one molecule Apaf-1 and one
molecule cytochrome c. Cytochrome c apparently docked between
the tandem -propellers of Apaf-1 at the rim of the disk. The hub
was thought to be formed by the CARD and part of the NOD [55]. A
subsequent EM reconstruction to 12.8 [30] allowed the placement
of individual subdomains of the X-ray model of the human Apaf-1
CARDNOD fragment [28] and of homology models of a six-bladed
and a seven-bladed -propeller. The resulting model dissects the
hub into an inner ring formed by the seven CARDS and an outer ring
formed by NBDHD1 subunits bridged by WHDs. A modeling study
of the Apaf-1 apoptosome complex challenged this view and presented a model in which the protomer arrangement resembles that
in AAA+ complexes [56]. Here, the hub is exclusively formed by subunits of the NODs, and the CARDs form a crown that sits on top. A recent cryo-EM study to 9.5 conrmed these results [31]. The NBDs
indeed form an inner ring reminiscent of the canonical AAA+ architecture, whereas an outer ring contains HD1 and WHDs in an alternating arrangement (Fig. 1). In the absence of procaspase-9 the CARDs are
disordered but together with procaspase-9 CARDs form a disk that is
exibly tethered to the apoptosome. Implications of these ndings
for caspase stimulation will be discussed in Section 6.
6. Caspase activation by the Apaf-1 apoptosome
Caspases are produced as zymogens, which are cleaved within
their catalytic domains to yield a large and a small subunit [57]. Apoptotic caspases are divided into two classes according to their position and function within the apoptotic pathway: initiator or effector
caspases [58]. Effector caspases are constitutive dimers lacking a prodomain, whereas initiator caspases possess a prodomain and are monomeric at physiological concentrations [57,58]. Effector caspases are
activated by cleavage of the interdomain linker allowing for rearrangement of the active site loops [59]. Although loop rearrangement
seems to be a general necessity for caspase activation, linker cleavage
is not a strict requirement for initiator caspase stimulation [60,61].
After several activator platforms for different initiator caspases had
been identied, two alternative models for initiator caspase activation
emerged: the proximity-induced dimerization model [15,62,63] and
the induced conformation model [6466]. The proximity-induced dimerization model is based on the observation that initiator caspase

1423

molecules are able to process and activate themselves if brought into

close contact to each other [35,6769]. It is thought that the active caspase species is a dimer where one monomer is catalytically active,
whereas the second monomer is inactive [70]. Catalytic competence is
achieved by rearrangement of the active site loops in the active monomer. In this view the respective activator platform, the Apaf-1 apoptosome in case of caspase-9, serves to achieve a local caspase
concentration higher than the dissociation constant for dimer formation
[62,63,71]. The induced conformation model assumes that the major determinant for caspase activation is not dimerization but an allosteric action of the activator platform on the caspase. This action induces a
conformational change of the caspase which in turn increases its activity. The nature of this conformational change is unknown and several
variants of this model are conceivable (reviewed in [64]).
The proximity-induced dimerization model is amongst others supported by a study using a chimeric caspase containing the CARD of
caspase-9 fused to the catalytic domain of caspase-8 [63]. The chimera
was able to restore the activation of executioner caspase in caspase-9
depleted cytosolic extracts supplied with exogenous dATP and cytochrome c to about 70% of the wild type level. Support for the induced
conformation model comes from the fact that a constitutively dimeric
caspase-9 mutant is only slightly more active than wild-type caspase9 and can not be activated by the apoptosome [65]. Further evidence
for the induced conformation model comes from a recent cryo-EM
study on the caspase-9 containing holo-apoptosome of Apaf-1 [72]. A
map calculated from holo-apoptosomes without symmetry restraints
only showed density for a single procaspase-9 molecule located on
the central hub. Moreover, the CARD disk assumes a tilted acentric orientation shifted away from the procaspase density. This suggests that
the acentric CARD disk might impose steric hindrance on procaspase
binding allowing only one catalytic procaspase-9 subunit to bind at a
time.
Unexpectedly, shortening of the procaspase prodomain linker by 14
or 24 residues severely compromises apoptosome dependent procaspase activity. If activation would proceed via proximity-induced dimerization, a short prodomain linker of 610 residues should still be long
enough to allow dimer assembly [73]. Moreover, there is evidence
from the EM-study that the prodomain linker is involved in binding of
procaspase-9 to the apoptosome [72]. This is well in line with the fact
that phosphorylation of caspase-9 at position T125 in the prodomain
linker leads to inhibition of apoptosis [7476]. It is tempting to speculate that the phosphorylation interferes with proper orientation of the
catalytic domain on the apoptosome. Taken together, these data argue
against a pure dimerization based activation mechanism but rather
point towards a proximity-induced association with a procaspase
monomer as the active species.
7. The Apaf-1 orthologs Dark and Ced-4
The components of apoptotic pathways have also been thoroughly studied in the nematode Caenorhabditis elegans and the fruity Drosophila melanogaster. The Drosophila Apaf-1 homolog Dark
(Drosophila Apaf-1 related killer) shares the same principal domain
structure as mammalian Apaf-1 [7779]. A cryo-EM reconstruction
to 6.9 of the Dark apoptosome shows structural similarities to
the Apaf-1 apoptosome, i.e. a wheel-like appearance where the
NODs form the hub and the WD40 tandem -propellers form the
rim of the wheel [80]. Different from the human apoptosome, the
Dark apoptosome contains eight instead of seven spokes, each consisting of one Dark monomer. To date, no crystal structure of autoinhibited Dark exists, but the structural similarity on the apoptosomal

Fig. 1. A. Subdomain structure of Apaf-1. Subdomains are color-coded, linkers are shown in gray. B. Conformational states of Apaf-1, the cartoon representation is color coded as in
1A. Top left: Apaf-1 in its autoinhibited state. Top right: Apaf-1 monomer in its assembly competent conformation. The yellow -propeller WD1 has adjusted its position to complete the binding site for cytochrome c between the inner faces of the two -propellers; the blue-turquoise NBDHD1 subunit is rotated to bare the oligomerization interfaces. Bottom: cartoon representation of the apoptosome (PDB code 3IZA). In the three states shown the CARD is not resolved [29,31] and is therefore not shown in the drawing.

1424

T.F. Reubold, S. Eschenburg / Cellular Signalling 24 (2012) 14201425

level suggests a similar mode of autoinhibition. However, there is

still a controversy concerning the role of cytochrome c in the activation process (reviewed in [81,82]). Although Drosophila cytochrome
c seems to play a role in certain developmental apoptotic situations
[8385], the bulk of the available data suggests that cytochrome c is
not required for apoptosome formation [8692]. This notion is supported by the in vitro reconstitution of Dark apoptosomes, which
does not require cytochrome c but occurs upon incubation with
unphysiologically high dATP concentrations in the presence of
EDTA [80,93].
In vivo, the caspase-9 homolog DRONC (Drosophila Nedd2-like
caspase) [92,94] can be activated by ablation of DIAP1 (Drosophila
inhibitor of apoptosis protein 1). DIAP1 is a y homolog of the mammalian IAPs (inhibitor of apoptosis proteins) and marks DRONC for
proteasomal degradation via its E3-ubiquitin ligase activity [95,96].
This suggests that Dark is constitutively active. However, another
study points to the existence of a yet unknown Dark activator [92].
So, the physiological regulation of Dark is far from clear and more
research is necessary to elucidate the chain of events that lead to
cell death in Drosophila.
Ced-4 (cell-death abnormality 4), the C. elegans homolog of
Apaf-1, possesses a CARD and an NOD, but no WRD. Unlike Apaf-1
Ced-4 is not autoinhibited but forms a complex with the Bcl-2 protein Ced-9 to maintain its inhibited state [97]. The crystal structure
of this complex reveals one molecule of Ced-9 bound to an asymmetric Ced-4 dimer [51]. There is no evidence for any mitochondrial
participation in the signaling cascade and consequentially cytochrome c is not needed for Ced-4 activation. Activation is achieved
by a third protein, Egl-1 (egg-laying defective 1), which binds to
Ced-9, thereby releasing the Ced-4 dimer from the inhibitory complex [98]. The sole nematode caspase, Ced-3, is activated by an octameric apoptosome assembled from four Ced-4 dimers [52]. While
the human and y apoptosomes display a at disk-like geometry,
the Ced-4 apoptosome possesses a funnel-like structure. The
CARDs of Ced-4 form two stacked tetramers, one of which contacts
the narrow end of the funnel that is formed by the NODs. Investigation of the mode of Ced-3 activation within the same study led to an
unexpected result. A soluble CARD-deleted Ced-3 fragment (residues 198503) could be stimulated in the presence of Ced-4 apoptosome [52]. This led to the hypothesis that the caspase binding
sites responsible for stimulation are located within the funnel. If
this were true, CARD interactions, which are indispensable for
other caspase activation platforms, would not be required for execution of nematode cell death. However, without further experiments employing full-length Ced-3 the proposed mechanism must
remain speculative.

8. Concluding remarks
The Apaf-1 apoptosome represents the central signaling hub
within the mitochondrial pathway of apoptosis. In recent years considerable information has been gained about both the architecture
and the function of apoptosomal complexes. Especially structural
analyses using X-ray crystallography and cryo electron microscopy
contributed substantially to the understanding of these molecular
switches. However, there are still open questions about important
aspects of the signal transduction process. The role of the nucleotide
exchange has yet to be determined and the mechanism of caspase
activation is even more subject of controversial discussion. Furthermore, it is still unknown how Apaf-1 interacts with regulating proteins other than cytochrome c. In recent years numerous interaction
partners of Apaf-1 have been identied, which inhibit or stimulate
apoptosome assembly. Detailed knowledge about these interactions, as future studies will certainly deliver, may be the key to target Apaf-1 for therapeutic purposes. The possibility to specically

modulate the function of Apaf-1 is highly desirable, given the central role of Apaf-1 in the mitochondrial pathway of apoptosis.
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