Molecular Cell, Vol.
9, 459–470, March, 2002, Copyright 2002 by Cell Press
Mechanisms of Caspase Activation
and Inhibition during Apoptosis
Yigong Shi1
Department of Molecular Biology
Princeton University
Lewis Thomas Laboratory
Washington Road
Princeton, New Jersey 08544
Caspases are central components of the machinery
responsible for apoptosis. Recent structural and bio-
chemical studies on procaspases, IAPs, Smac/DIA-
BLO, and apoptosome have revealed a conserved
mechanism of caspase activation and inhibition. This
article reviews these latest advances and presents our
current understanding of caspase regulation during
apoptosis.
Introduction
Apoptosis, or programmed cell death, plays a central
role in the development and homeostasis of all multi-
cellular organisms (Horvitz, 1999; Jacobson et al., 1997).
In humans, both excessive and insufficient apoptosis
can lead to severe pathological consequences. Sup-
pression of the apoptotic machinery causes autoim-
mune diseases and is a hallmark of cancer (Hanahan
and Weinberg, 2000; Thompson, 1995). For example,
the apoptotic protease-activating factor 1 (Apaf-1) is
frequently inactivated in cancers such as malignant mel-
anoma (Soengas et al., 2001). On the other hand, abnor-
mal upregulation of apoptosis contributes to neurologi-
cal disorders (Yuan and Yankner, 2000).
The critical involvement of a cysteine protease CED-3
in apoptosis was first discovered in the nematode
Caenorhabditis elegans 8 years ago (Yuan et al., 1993).
Since then, compelling evidence has demonstrated that
the mechanism of apoptosis is evolutionarily conserved,
executed by a family of cysteine proteases that cleave
after an Asp residue in their substrates, hence the name
caspase (Alnemri et al., 1996). At least 14 distinct mam-
malian caspases have been identified, with their or-
thologs present in species ranging from the nematode
to the dipteran Drosophila melanogaster and the lepi-
dopteran Spodoptera frugiperda. Although the first
mammalian caspase, caspase-1 or ICE (interleukin
1␤-converting enzyme), was identified to be an impor-
tant regulator of inflammatory response, at least 8 of
the 14 caspases play important roles during apoptosis
(Budihardjo et al., 1999; Earnshaw et al., 1999; Fesik
and Shi, 2001; Salvesen and Dixit, 1997; Thornberry and
Lazebnik, 1998).
Caspases involved in apoptosis are generally divided
into two categories, the initiator caspases, which include
caspase-2, -8, -9, and -10, and the effector caspases,
1
Correspondence: yshi@molbio.princeton.edu
Review
which include caspase-3, -6, and -7 (Figure 1). An initia-
tor caspase is characterized by an extended N-terminal
prodomain (⬎90 amino acids) important for its function,
whereas an effector caspase contains 20–30 residues
in its prodomain sequence. All caspases are produced
in cells as catalytically inactive zymogens and must un-
dergo proteolytic activation during apoptosis. The acti-
vation of an effector caspase (such as caspase-3 or -7)
is performed by an initiator caspase (such as caspase-9)
through cleavage at specific internal Asp residues that
separate the large and small subunits (Figure 1). The
initiator caspases, however, are autoactivated. As the
activation of an initiator caspase in cells inevitably trig-
gers a cascade of downstream caspase activation, it is
tightly regulated and often requires the assembly of a
multicomponent complex under apoptotic conditions.
For example, the activation of procaspase-9 is facili-
tated by Apaf-1 and cytochrome c (Cyt. c) (Li et al.,
1997), which form an ⵑ1.4 MDa complex dubbed an
apoptosome in the presence of dATP or ATP (Rodriguez
and Lazebnik, 1999; Saleh et al., 1999; Zou et al., 1999).
Once activated, the effector caspases are responsible
for the proteolytic cleavage of a broad spectrum of cellu-
lar targets, leading ultimately to cell death. The known
cellular substrates include structural components (such
as actin and nuclear lamin), regulatory proteins (such
as DNA-dependent protein kinase), inhibitors of deoxyri-
bonuclease (such as DFF45 or ICAD), and other proapo-
ptotic proteins and caspases. Cleavage of the DFF45
(DNA fragmentation factor 45) leads to removal of its
inhibition of DFF40 or CAD (caspase-activated deoxyri-
bonuclease), which degrades the chromosomes into
nucleosomal fragments during apoptosis (Enari et al.,
1998; Liu et al., 1997, 1998).
Caspases are specific proteases, recognizing at least
four contiguous amino acids, named P4-P3-P2-P1, and
cleaving after the C-terminal residue (P1), usually an
Asp. Although the P1 residue was thought to be exclu-
sively Asp, recent studies indicate that some caspases
can also cleave after Glu (Hawkins et al., 2000; Sriniva-
sula et al., 2001). Interestingly, the preferred P3 position
is invariantly Glu for all mammalian caspases examined
(Thornberry et al., 1997). Thus the preferred specificity
of cleavage for caspases can be described as X-Glu-X-
Asp.
Structural characterization of caspase-1 reveals a ho-
modimeric structure (Walker et al., 1994; Wilson et al.,
1994). It is now believed that the functional caspase unit
is a homodimer, with each monomer comprising a large
(20 kDa) and a small (10 kDa) subunit. Homodimerization
is mediated by hydrophobic interactions, with 6 antipar-
allel ␤ strands from each catalytic subunit forming a
single contiguous 12-stranded ␤ sheet (Figure 2A). Sev-
eral ␣ helices and short ␤ strands are located on either
side of the central ␤ sheet, giving rise to a globular fold.
The active sites, formed by four protruding loops from
the scaffold, are located at two opposite ends of the ␤
sheet (Figure 2A). In addition to caspase-1, structural
information is available for caspase-3 (Mittl et al., 1997;
Rotonda et al., 1996), caspase-7 (Wei et al., 2000), cas-
Molecular Cell
460
pase-8 (Blanchard et al., 1999; Watt et al., 1999), and
more recently, caspase-9 (Renatus et al., 2001). Rather
than giving a general review on the structural biology
of apoptosis (Fesik, 2000; Shi, 2001), this article focuses
on the latest developments and surveys our current un-
derstanding of caspase regulation at a molecular level.
Conserved Features of the Active Site
The substrate-binding groove is shaped by four sur-
rounding loops, named L1, L2, L3, and L4 (Figure 2B).
The L1 loop constitutes one side of the groove whereas
L4 represents the other side (Figure 2C). Loop L3 and
the following ␤ hairpin, collectively referred to as L3,
are located at the base of the groove. Loop L2, which
harbors the catalytic residue Cys, is positioned at one
end of the groove with the side chain of Cys pointing
along the groove, poised for binding and catalysis.
Among these four loops, L1 and L3 exhibit a relatively
conserved length as well as composition among all
mammalian caspases (Figure 1). In contrast, L2 and L4
are highly divergent. These four loops determine the
sequence specificity of the substrates. The binding
pockets for the P4-P3-P2-P1 residues of the substrate
are named S4-S3-S2-S1 subsites, respectively. Struc-
tural studies using covalent peptide inhibitors reveal
that these pockets are located mostly between the base
(L3) and the two sides (L1 and L4).
Figure 1. Schematic Diagram of the Mamma-
lian Caspases
Except caspase-11 (mouse), -12 (mouse),
and -13 (bovine), all listed caspases are of
human origin. Their phylogenetic relationship
(left) appears to correlate with their function
in apoptosis or inflammation. The initiator and
effector caspases are labeled in purple and
red, respectively. The position of the first acti-
vation cleavage (between the large and small
subunits) is highlighted with a large arrow
while additional sites of cleavage are repre-
sented by medium and small arrows. In con-
trast to other protease zymogens, removal of
the prodomain of a caspase is unnecessary
for its catalytic activity. The four surface loops
(L1-L4) that shape the catalytic groove are
indicated. The catalytic residue Cys is shown
as a red line at the beginning of loop L2. This
diagram is scaled according to the lengths
of caspases and the location of functional
segments.
All known caspases share a similar conformation at
the substrate-binding groove (Figure 2B). The S1 and
S3 sites are nearly identical among all caspases whereas
the location of the S2 and S4 sites is highly conserved.
The P1 residue (Asp) is coordinated by three invariant
residues at the S1 site, an Arg from L1, a Gln at the
beginning of L2, and an Arg at the end of L3 (Earnshaw
et al., 1999). The Arg residue on the L3 loop also coordi-
nates the P3 residue (Glu). The S2 and S4 sites map
mainly to the L3 and L4 loops; thus the P2 and P4
residues exhibit greater sequence variation. For exam-
ple, the L4 loop in caspase-1, -8, or -9 is considerably
shorter than that in caspase-3 or -7, resulting in a shal-
lower substrate-binding groove (Figure 2D). This obser-
vation is consistent with a bulky hydrophobic residue
as the preferred P4 residue for caspase-1, -8, or -9.
The conformational similarity at the active site is ex-
tended to surrounding structural elements. Most nota-
bly, the loops L4 and L2 from one catalytic subunit are
stabilized by the N terminus (loop L2⬘) of the small sub-
unit of the other catalytic subunit (Figure 2), forming the
so-called “loop bundle” (Chai et al., 2001a).
Activation Mechanisms of the Effector Procaspases
Except procaspase-9, all other procaspase zymogens
are inactive and require proteolytic activation. Given the
presence of the four active site loops in the zymogen,
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461

Figure 2. Structural Features of Caspases

(A) Representative structure of an inhibitor-
bound caspase-3 (PDB code 1DD1). The
small and large subunits are colored orange
and blue, respectively. The bound peptide in-
hibitor is shown in pink. The four surface
loops that constitute the catalytic groove of
one heterodimer are labeled. The apostrophe
denotes the other heterodimer. Note that L2⬘
stabilizes the active site of the adjacent het-
erodimer.
(B) The active site conformation of all known
caspases is conserved. Of the four loops, L1
and L3 are relatively constant while L2 and
L4 exhibit greater variability. The catalytic
residue Cys is highlighted in red.
(C) Schematic diagram of the substrate-bind-
ing groove. L1 and L4 constitute two parallel
sides of the groove while L3 serves as the
base. L2, harboring the catalytic residue Cys,
is positioned at one end of the groove, poised
for catalysis. L2⬘ plays a critical role by stabi-
lizing the conformation of the L2 and L4
loops.
(D) Conformation of the L4 loop in five known
caspase structures. The L4 loop dictates the
P4 specificity of the substrate. Short L4 loops
(caspase-1, -8, and -9) allow bulkier and hy-
drophobic residue in the P4 position whereas
extended L4 loops (caspase-3 and -7) prefer
Asp. Coloring scheme is the same as (B). Fig-
ures were prepared using MOLSCRIPT
(Klaulis, 1991) and GRASP (Nicholls et al.,
1991).

it was intuitively unclear why this is the case. The recent
crystal structure of procaspase-7 (Figure 3A) provides
an explanation to this puzzle (Chai et al., 2001a; Riedl
et al., 2001a).
Compared to the inhibitor-bound caspase-7, the core
structural elements of the procaspase-7 zymogen are
nearly identical, with less than 0.8 Å root-mean-square-
deviation for all aligned C␣ atoms. However, three of
the four active site loops adopt drastically different con-
formations. L3, which forms the base of the catalytic
groove in the active caspase-7, is unraveled and loos-
ened above the base (Figure 3C). Loop L4, which forms
one side of the catalytic groove in the active caspase-7,
is located farther away from L3, flattening the active site

Figure 3. Mechanisms of Procaspase-7 Activation and Substrate Binding

(A) Structure of a procaspase-7 zymogen (PDB code 1K86). Compared to that of the inhibitor-bound caspase-7, the conformation of the active
site loops does not support substrate binding or catalysis. The L2⬘ loop, locked in a closed conformation by covalent linkage, is occluded
from adopting its productive and open conformation.
(B) Structure of an active and free caspase-7 (PDB code 1K88). The active site loops are still flexible. Despite an interdomain cleavage, the
L2⬘ loop still exists in the closed conformation, indicating an induced-fit mechanism for binding to inhibitors/substrates.
(C) Comparison of the conformation of the active site loops. Compared to the procaspase-7 zymogen or the free caspase-7, the L2⬘ loop is
flipped 180o in the inhibitor-bound caspase-7 to stabilize loops L2 and L4.
Molecular Cell
462
pocket. Most importantly, loop L2, which contains the
catalytic residue Cys186, is rotated 90⬚, making this resi-
due inaccessible to solvent. Among the four loops, only
L1 retains its active site conformation.
These rearrangements in the procaspase-7 zymogen
do not allow formation of a substrate-binding groove.
In particular, the loop bundle seen in the inhibitor-bound
caspases is missing in the procaspase-7 zymogen as
the L2⬘ loop is flipped by 180⬚, existing in a “closed”
conformation (Figure 3C). These structural rearrange-
ments explain why the procaspase-7 zymogen does not
possess detectable catalytic activity.
What makes the active site of the procaspase-7 zymo-
gen so different from that of the inhibitor-bound cas-
pase-7? One essential reason is that the unprocessed
nature of the procaspase-7 zymogen locks the L2⬘ loop
in the closed conformation and occludes it from forming
a loop bundle with the L2 and L4 loops. This inability
can only be removed by a proteolytic cleavage after
Asp198, which allows L2⬘ to switch to its open conforma-
tion as observed in the inhibitor-bound caspase-7. As
the interactions that stabilize the loop bundle are gener-
ally conserved among caspases, this mechanism is
likely to be true for other caspases.
In this mechanism, the ability of L2⬘ to move freely
in response to inhibitor/substrate binding is a decisive
feature. In caspase-7, this ability is acquired through
proteolytic cleavage after Asp198. But, because L2⬘ is
at the N terminus of the small subunit, inverting the order
of primary sequences of the large and small subunits
could free L2⬘ and hence constitutively activate cas-
pases. Indeed, this prediction was confirmed for cas-
pase-3 and -6 (Srinivasula et al., 1998b) as well as for
the Drosophila caspase drICE (Wang et al., 1999).
Why does procaspase-9 exhibit a basal level of activ-
ity prior to proteolytic activation (Stennicke et al., 1999)?
The answer may lie in the fact that caspase-9 contains
an expanded L2 loop, which could allow enough confor-
mational flexibility for the L2⬘ loop to move to its produc-
tive conformation without an interdomain cleavage.
Mechanisms of Substrate Binding
Because the active sites of all inhibitor-bound caspases
are highly similar (Figure 2B), it was once thought that
the substrate-binding grooves on caspases are pre-
formed. However, in the structure of the free caspase-7
(Chai et al., 2001a), these loops are flexible and quite
different from those in the inhibitor-bound caspase-7
(Figure 3B). Surprisingly, despite activation cleavage,
the L2’ loop in the isolated active caspase-7 still exists
in the closed conformation, mimicking the procaspase-7
zymogen (Figure 3C). Consequently, the loop bundle is
not properly assembled and the catalytic cleft is not well
defined.
This unanticipated finding indicates that the isolated
active caspase-7 adopts a conformation intermediate
between that of the zymogen and that of the inhibitor-
bound caspase-7. Thus inhibitor/substrate binding trig-
gers a 180⬚ flipping of the L2⬘ loop in the active
caspase-7, changing its conformation from a closed to
an open form (Figure 3C). Each cycle of substrate bind-
ing and catalysis is a process of induced fit, accompa-
nied by the back-and-forth flipping of the L2⬘ loop. This
observation argues that the substrate-bound state is
transient and can be trapped by the covalent peptide
inhibitors. It remains to be seen whether this mechanism
is generally applicable to other caspases.
Inhibition of Caspases by IAPs
Caspases are regulated by many cellular processes.
For example, caspases are subject to transcriptional
regulation and posttranslational modification (Earnshaw
et al., 1999). In addition, active caspases can be perma-
nently eliminated by the ubiquitination-mediated protea-
some degradation pathway (Huang et al., 2000; Suzuki
et al., 2001b).
The enzymatic activity of caspases is subject to inhibi-
tion by the conserved IAP (inhibitor of apoptosis) family
of proteins (Deveraux and Reed, 1999; Hay, 2000). The
IAPs, originally identified in the genome of baculovirus
based on their ability to suppress apoptosis in infected
host cells, antagonize cell death by interacting with and
inhibiting the enzymatic activity of mature caspases.
Eight distinct mammalian IAPs, including XIAP, c-IAP1,
c-IAP2, and ML-IAP/Livin (Ashhab et al., 2001; Kasof
and Gomes, 2001; Vucic et al., 2000), have been identi-
fied, and they target the initiator caspase, caspase-9,
and the effector caspases, caspase-3 and -7 (Deveraux
and Reed, 1999). These IAP proteins do not inhibit other
caspases, such as caspase-6 or -8.
The functional unit in IAPs is the baculoviral IAP repeat
(BIR), which contains ⵑ80 amino acids folded around a
zinc atom. XIAP, c-IAP1, and c-IAP2 contain three BIR
domains each, with the different BIR domains exhibiting
distinct functions. In these IAPs, the third BIR domain
(BIR3) potently inhibits the activity of processed cas-
pase-9 whereas the linker region between BIR1 and
BIR2 selectively targets caspase-3 and -7 (Fesik and
Shi, 2001; Shi, 2001). This inhibition is highly specific;
for example, BIR3 exhibits no inhibition of caspase-3
even at elevated concentrations whereas the linker re-
gion between BIR1 and BIR2 has no impact on caspase-
9. Mutational analysis revealed that, in the XIAP-linker-
BIR2 fragment, substitution of Asp148 or Leu141 to Ala
nearly abolished caspase-3 inhibition (Sun et al., 1999).
The corresponding BIR domains in XIAP, c-IAP1, and
c-IAP2 are highly conserved in both sequence and func-
tion. For example, BIR3 of XIAP is more similar to BIR3
of c-IAP1 or c-IAP2 than to BIR2 or BIR1 of XIAP. In
contrast to these IAPs, ML-IAP/Livin, which is highly
expressed in melanoma and lymphoma, contains only
a single BIR domain but was reported to inhibit both
caspase-3 and -9 (Ashhab et al., 2001; Kasof and
Gomes, 2001; Vucic et al., 2000). Another single BIR-
containing IAP, survivin, does not inhibit caspase activ-
ity in vitro. In contrast to the relatively stable expression
levels of other IAPs, expression of survivin oscillates
with cell cycle and peaks at the G2/M phase (Li et al.,
1998). Survivin appears to play an important role in mito-
sis (Altieri, 2001; Li et al., 1998).
Mechanisms of IAP-Mediated Inhibition
of Effector Caspases
Recent structural analyses reveal the precise mecha-
nism of IAP-mediated inhibition of caspase-3 and -7
(Chai et al., 2001b; Huang et al., 2001; Riedl et al., 2001b).
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463
In the structures, the linker peptide N-terminal to XIAP-
BIR2 forms highly similar interactions with both cas-
pase-3 and -7 (Figure 4A). Compared to the covalent
peptide inhibitors, the linker segment of XIAP occupies
the active site of caspases in a reverse orientation, re-
sulting in a blockade of substrate entry (Figure 4B).
Asp148 of XIAP, which was shown to be critical for the
inhibition of caspase-3, binds the S4 pocket in the same
manner as the P4 residue of the covalent peptide inhibi-
tors. In addition, Val146 makes a similar set of van der
Figure 4. Mechanisms of IAP-Mediated Inhi-
bition of Effector Caspases and p35-Medi-
ated Pan-Caspase Inhibition
(A) Superposition of the structures of cas-
pase-3 (brown) and -7 (green) together with
their bound XIAP fragments colored blue and
pink, respectively. The interactions primarily
occur between a linker segment N-terminal
to the BIR2 domain of XIAP and the active
site of caspase-3 or -7 (boxed region). The
small red circle on BIR2 marks where the
Smac N-terminal tetrapeptide binds and the
large red circle indicates a likely second inter-
face with Smac.
(B) Close-up view of the active sites of cas-
pase-3 and -7 bound to their respective XIAP
fragments. Two hydrophobic residues of
XIAP, Leu141 and Val146, make multiple van
der Waals interactions with a conserved hy-
drophobic pocket on caspase-3 or -7. Asp148
of XIAP, occupying the S4 pocket, hydrogen
bonds to neighboring residues in caspase-3
or -7.
(C) Close-up view of the covalent inhibition
of caspase-8 by p35. The thioester intermedi-
ate is shown between Asp87 of p35 and
Cys360 (active site residue). The N terminus
of p35 restricts solvent access to this inter-
mediate.
Waals contacts to surrounding caspase residues as
does the P2 residue. In contrast to the covalent peptide
inhibitors, Gly144 is located close to the S1 pocket,
within van der Waals contact distance of the catalytic
Cys in the caspases.
Interestingly, although the linker sequence between
BIR1 and BIR2 of XIAP plays a dominant role in inhibiting
caspase-3 and -7, this fragment in isolation is insufficient
(Chai et al., 2001b; Sun et al., 1999). However, an engi-
neered protein with the linker peptide fused either N-
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464
or C-terminal to BIR1 was fully able to bind and inhibit
caspase-3 while neither BIR1 nor BIR2 in isolation exhib-
ited any effect (Sun et al., 1999). These observations
suggest that the linker peptide needs to be presented
in a “productive” conformation by a surrounding BIR
domain. In support of this hypothesis, the linker peptide
fused to glutathione S-transferase (GST) was able to
inhibit caspase-3 and -7 (Chai et al., 2001b; Huang et
al., 2001). Nevertheless, the BIR domains also contribute
to the inhibition of caspases, as XIAP exhibits about 20-
fold higher potency than the GST-linker peptide fusion
(Chai et al., 2001b; Huang et al., 2001). Consistent with
this observation, XIAP-BIR2 also makes direct contacts
to caspase-3 in the crystal structure (Riedl et al., 2001b).
More importantly, the BIR domains provide a scaffold for
Smac/DIABLO binding in order to relieve IAP-mediated
inhibition of the effector caspases (see below).
Mechanisms of IAP-Mediated Inhibition
of Caspase-9
Only processed caspase-9 is subject to inhibition by
IAPs. Although both BIR2 and BIR3 of XIAP can inhibit
caspase-9, BIR3 displays tighter binding affinity and
higher potency. Mutation of Trp310 or Glu314 in BIR3
completely abrogates XIAP-mediated inhibition of cas-
pase-9, while amino acids outside of BIR3 are unneces-
sary for this inhibition (Sun et al., 2000). In the structure,
these two residues are located close to each other,
suggesting that this region is involved in binding and
inhibiting caspase-9. Indeed, BIR3 of XIAP binds to the
N terminus of the small subunit of caspase-9 that be-
comes exposed after proteolytic processing (Srinivasula
et al., 2001). This binding presumably brings BIR3 close
to the active site of caspase-9, which may block sub-
strate entry and subsequent catalysis. In support of this
hypothesis, when the N-terminal four residues of the
caspase-3 small subunit were replaced with those from
caspase-9, the resulting protein could be inhibited by
BIR3 (Srinivasula et al., 2001).
The interaction between the N-terminal four residues
of the caspase-9 small subunit and XIAP-BIR3 is neces-
sary but may not be sufficient for the XIAP-mediated
inhibition. Mutation of His343 in XIAP-BIR3, which is
located in a different area than Trp310 and Glu314, re-
sults in a loss of caspase-9 inhibition, suggesting that
His343 also makes important contacts to caspase-9
(Sun et al., 2000).
Covalent Inhibition of Caspases by p35
IAPs are not the only natural inhibitors to caspases. In
contrast to XIAP, which only affects caspase-3, -7, and
-9, the baculoviral p35 protein is a pan-caspase inhibitor,
and it potently targets most caspases both in vivo and in
vitro (Miller, 1999). Caspase inhibition by p35 correlates
with the cleavage of its reactive site loop after Asp87,
which leads to the translocation of the N terminus of
p35 into the active site of caspases (Bump et al., 1995;
Zhou et al., 1998). The crystal structure of caspase-8
in complex with p35 reveals that the catalytic residue
Cys360 of caspase-8 is covalently linked to the Asp87 of
p35 through a thioester bond (Xu et al., 2001). Although a
thioester bond is generally susceptible to hydrolysis,
this bond is protected by the neighboring N terminus of
p35, which occludes access by water molecules (Figure
4C). Another protein, the serpin CrmA derived from the
cowpox virus, can also inhibit several caspases, likely
through similar covalent modification. This unique cova-
lent mechanism adds to the complexity of caspase inhi-
bition by natural proteins. It is important to note, how-
ever, that an equivalent of p35 in mammals has not been
identified.
Function of Smac/DIABLO
During apoptosis, the IAP-mediated caspase inhibition
is removed by a mitochondrial protein named Smac
(second mitochondria-derived activator of caspases)
(Du et al., 2000) or DIABLO (direct IAP-binding protein
with low pI) (Verhagen et al., 2000). Upon stimulation of
apoptosis, Smac/DIABLO is released from the inter-
membrane space of mitochondria into the cytosol, to-
gether with Cyt. c. Whereas Cyt. c directly activates
Apaf-1 and caspase-9, Smac/DIABLO interacts with
multiple IAPs and removes IAP-mediated inhibition of
both initiator and effector caspases (Chai et al., 2000;
Srinivasula et al., 2000).
Smac functions as an elongated dimer, spanning over
130 Å in length (Chai et al., 2000) (Figure 5A). The wild-
type Smac protein binds to both the BIR2 and the BIR3
domains of XIAP but not the BIR1 domain. In contrast,
the monomeric Smac mutants interact strongly with
BIR3 but do not form a stable complex with BIR2 (Chai
et al., 2000). Because the linker sequence immediately
preceding BIR2 is involved in binding and inhibiting cas-
pase-3, Smac monomers cannot relieve the IAP-medi-
ated caspase-3 inhibition. Despite the ability to interact
with BIR3, the monomeric Smac mutants are less able
to relieve IAP-mediated caspase-9 inhibition (Chai et al.,
2000).
The N-terminal mitochondria-targeting sequence of
Smac is proteolytically removed upon import (Du et al.,
2000). These freshly exposed N-terminal residues play
an indispensable role in Smac function; a seven residue
peptide including these residues can remove the XIAP-
mediated caspase-9 inhibition. Strikingly, a missense
mutation of the N-terminal residue Ala to Met in Smac
leads to a complete loss of interactions with XIAP and
the concomitant loss of Smac function (Chai et al., 2000).
A Tetrapeptide IAP-Binding Motif
The molecular explanation for the indispensable role of
the Smac N-terminal sequences is provided by struc-
tures of XIAP-BIR3 bound to either a monomeric Smac
protein (Wu et al., 2000) or a nine residue Smac peptide
(Liu et al., 2000). The Smac N-terminal tetrapeptide (Ala-
Val-Pro-Ile) recognizes an acidic surface groove on
BIR3, with the first residue Ala binding a hydrophobic
pocket and making hydrogen bonds to neighboring
XIAP residues (Figure 5B). The next three residues also
interact with surrounding hydrophobic residues in BIR3.
To accommodate these interactions, the N terminus of
Smac must be free, thus explaining why only the mature
Smac can bind to the IAPs. Modeling studies indicate
that replacement of Ala by a bulkier residue will cause
steric hindrance while Gly substitution may result in an
entropic penalty as well as loss of binding in the hy-
drophobic pocket. This analysis explains why mutation
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465
of Ala to Met or Gly abrogated interactions with the BIR
domains. Since most of the important interface residues
of BIR3 are conserved in BIR2, the isolated N-terminal
Smac peptides can also interact with the BIR2 domain
of XIAP, though with a lower binding affinity (Liu et al.,
2000).
In fruit flies, the anti-death IAP, DIAP1, binds and
inhibits several Drosophila caspases (Hay, 2000). This
function is countered by the proapoptotic proteins,
Reaper, Grim, Hid, and more recently, Sickle (Christich
et al., 2002; Srinivasula et al., 2002; Wing et al., 2002),
which physically interact with the BIR2 domain of DIAP1
and remove its inhibitory effect on caspases. Thus
Reaper, Grim, Hid, and Sickle are functional homologs
of the mammalian protein Smac/DIABLO. Supporting
this notion, the N-terminal tetrapeptides of Reaper,
Grim, Hid, and Sickle closely resemble that of the Smac/
DIABLO (Figure 5C). The Smac-binding surface groove
on XIAP-BIR3 comprises highly conserved residues
among the BIR3 domains of c-IAP1 and c-IAP2 and the
BIR2 domain of DIAP1, suggesting a conserved mode
of interactions. Indeed, the crystal structures of DIAP1-
BIR2 by itself and in complex with the N-terminal pep-
Figure 5. A Conserved IAP-Binding Tetra-
peptide Motif
(A) Structure of the mature Smac. The disor-
dered N-terminal residues are shown as dot-
ted lines.
(B) Close-up view of the Smac N-terminal tet-
rapeptide binding to the BIR3 surface groove.
The BIR3 domain is shown either by electro-
static potential (left panel) or in ribbon dia-
gram (right panel) to highlight the interac-
tions. The amino and carbonyl groups of the
N-terminal Ala make several hydrogen bonds
to conserved residues in XIAP.
(C) A conserved motif of IAP-binding tetra-
peptides. The tetrapeptide motif has the con-
sensus sequence A-(V/T/I)-(P/A)-(F/Y/I/V/S).
The Drosophila proteins have an additional
binding component (conserved 6th–8th res-
idues).
(D) A conserved IAP-binding mode from
mammals to fruit flies. The structure of
DIAP1-BIR2 is superimposed with that of the
XIAP-BIR3 domain, with their corresponding
bound peptides Hid (pink), Grim (blue), and
Smac/DIABLO (green).
tides from Grim, Hid, and Sickle reveal that the binding
of these N-terminal tetrapeptides precisely match that
of the Smac-XIAP interactions (Srinivasula et al., 2002;
Wu et al., 2001) (Figure 5D). For Grim and Hid, the next
three conserved residues also contribute to DIAP1 bind-
ing through hydrophobic interactions.
Using a direct XIAP-binding assay, another mamma-
lian protein, HtrA2/Omi, was recently identified by five
independent groups (Hedge et al., 2002; Martin et al.,
2002; Suzuki et al., 2001a; van Loo et al., 2002; Verhagen
et al., 2002). Similar to Smac, HtrA2/Omi is believed to
be a resident protein of the intermembrane space of
mitochondria and is released to the cytosol during apo-
ptosis. The mature form of HtrA2/Omi contains three
functional segments, an IAP-binding tetrapeptide motif
at its N terminus, a central serine protease domain, and a
PDZ domain at its C terminus. Interestingly, the catalytic
activity of the serine protease domain is absolutely es-
sential to the HtrA2/Omi-mediated cell killing activity
whereas the N-terminal IAP-binding motif appears less
critical. HtrA2/Omi is unlikely to induce cell death
through a nonspecific protease activity as it appears to
exhibit stringent substrate specificity (Gray et al., 2000).
Molecular Cell
466

Figure 6. Schematic Diagram of the Mechanisms of Caspase Activation and Inhibition

(Left box) The tetrapeptide motif (red arrows), present in the N termini of Smac and the small subunit of caspase-9, binds to a surface groove
on the BIR domains of IAPs (XIAP, c-IAP1, c-IAP2, and Livin/ML-IAP). This motif is responsible for both caspase-9 inhibition through binding
BIR3 and the relief of inhibition through displacement of the caspase-9 tetrapeptide with a Smac tetrapeptide. Positive feedback cleavage
by caspase-3 or -7 permanently removes IAP-mediated inhibition of caspase-9. The released caspase-9 linker peptide can antagonize IAP-
mediated inhibition of other caspases.
(Right box) On the other hand, a linker segment N-terminal to the BIR2 domain of XIAP is primarily responsible for caspase-3 inhibition. Smac
binding to the BIR2 domain may make this linker segment unavailable for binding to caspase-3. For clarity, only BIR domains but not the full-
length IAPs are shown in this figure.

Since PDZ domains typically interact with the C-terminal
hydrophobic residues of membrane receptors and ion
channels (Harris and Lim, 2001), the PDZ domain in
HtrA2/Omi may further regulate its protease activity. The
precise mechanism of HtrA2/Omi-mediated apoptosis
remains elusive.
Removal of Caspase Inhibition by Smac/DIABLO
During apoptosis, Smac/DIABLO reactivates the pro-
cessed initiator as well as effector caspases through
distinct mechanisms, although both require physical in-
teractions with IAPs. The N terminus (Ala-Thr-Pro-Phe)
of the small subunit of caspase-9 was found to contain
an IAP-binding tetrapeptide motif (Srinivasula et al.,
2001). Subsequent experiments confirmed that this se-
quence is indeed primarily responsible for the interac-
tions between the processed caspase-9 and XIAP (Srini-
vasula et al., 2001). In the absence of proteolytic
processing, procaspase-9 is unable to interact with
IAPs. Proteolytic processing of procaspase-9 at Asp315
leads to the exposure of an internal tetrapeptide motif,
which recruits IAPs to inhibit caspase-9 (Figure 6). The
mature Smac binds IAPs, again using a similar N-ter-
minal tetrapeptide. Thus, a conserved IAP-binding motif
in caspase-9 and Smac mediates opposing effects on
caspase activity (Figure 6).
During apoptosis, caspase-9 can be further cleaved
after Asp330 by downstream caspases such as cas-
pase-3 (Figure 6). This positive feedback not only perma-
nently removes XIAP-mediated caspase-9 inhibition but
also releases a 15 residue peptide that is able to relieve
IAP-mediated inhibition of other caspases. This mecha-
nism ensures that less than a stoichiometric amount of
Smac can remove the IAP-mediated caspase-9 inhibi-
tion as transient activation of caspase-9 may lead to the
activation of caspase-3 and ensuing positive feedback.
Although the Smac tetrapeptide in isolation can re-
move IAP-mediated caspase-9 inhibition, it plays a less
direct role in the removal of IAP-mediated inhibition of
effector caspases (Figure 6). The binding site for this
tetrapeptide motif maps to the surface of BIR2 or BIR3,
whereas the fragment responsible for inhibiting cas-
pase-3 or -7 is located between BIR1 and BIR2 of XIAP.
Although a conclusive mechanism remains elusive,
modeling studies of a Smac/BIR2/caspase-3 complex
suggest that steric clashes preclude XIAP-BIR2 from
simultaneously binding to caspase-3 and Smac (Chai et
al., 2001b). In this model, binding to the BIR2 domain
requires not only the N-terminal tetrapeptide of Smac
but also an extensive surface available only in the wild-
type dimeric Smac protein. This model is consistent
with the observation that monomeric Smac mutants only
Review
467
weakly interacted with BIR2 and were unable to remove
the IAP-mediated caspase-3 inhibition (Chai et al., 2000).
Induced Proximity Model for the Initiator Procaspases
The initiator caspases invariably contain one of two pro-
tein-protein interaction motifs, the CARD (caspase re-
cruitment domain) or the DED (death effector domain).
These motifs interact with similar motifs present on
oligomerized adapter proteins, thus bringing multiple
initiator caspase molecules into close proximity with one
another and presumably facilitating their autoactivation.
This hypothesis is summarized as the induced-proximity
model (Salvesen and Dixit, 1999). For example, procas-
pase-8 contains two copies of the DED, which interact
with the DED of the adapter protein FADD (Fas-associ-
ated death domain). FADD, in turn, is a component of
the multimeric death-inducing signaling complex (DISC)
at the cell membrane (Ashkenazi and Dixit, 1998). Through
association with FADD, procaspase-8 is brought into
the DISC, resulting in its autocleavage and activation.
Another well-characterized paradigm involves pro-
caspase-9 activation. During apoptosis, Cyt. c is re-
leased from mitochondria into the cytoplasm, where it
binds to Apaf-1 and, in the presence of dATP or ATP,
assembles into a complex called the apoptosome (Saleh
et al., 1999; Zou et al., 1999). The primary function of
the apoptosome is to recruit procaspase-9 through a
CARD-CARD interaction between caspase-9 and Apaf-1
and facilitate autoactivation of caspase-9. Interestingly,
Apaf-1 is a member of a conserved family of adaptor
proteins that contain an N-terminal CARD, a central nu-
cleotide-binding oligomerization domain (NOD), and a
C-terminal sensing domain for intracellular ligands (Ino-
hara and Nunez, 2001). Members of the NOD family also
include Ipaf (Poyet et al., 2001), Nod1 (Inohara et al.,
1999), and Nod2 (Ogura et al., 2001). The caspase-acti-
vating mechanisms by these NODs are likely to be con-
served as well. Interestingly, the NOD proteins in animals
Figure 7. Structure of Caspase-9 and the
Apoptosome
(A) Two views of the heptameric apoptosome
comprising Apaf-1, Cyt. c, and dATP. The
CARD domains are located in the central hub
while the WD40 repeats were mapped to the
end of the spoke.
(B) Structure of the inhibitor-bound caspase-
9. Only half of the caspase-9 dimer contains
a well-formed active site (shown in green),
covalently bound by an inhibitor. The active
site loops in the other half, shown in pink, do
not form a functional catalytic groove.
(C) A proposed model for the activation of
caspase-9. The apoptosome-bound cas-
pase-9 exists in an inactive conformation.
The high local concentrations of caspase-9
within the apoptosome drive the recruitment
of additional caspase-9 monomers, which are
activated upon dimerization.
closely resemble a large family of disease-resistant
genes in plants (Dangl and Jones, 2001).
The induced proximity model appears to be consis-
tent with a number of observations. For example, bacte-
rially expressed caspases are almost always processed
to their mature forms, presumably due to high local
concentrations. In mammalian cells, forced oligomeriza-
tion of procaspase-8 through the use of FKBP (FK506
binding protein) and FK1012 (a dimeric form of FK506)
led to activation of procaspase-8 and subsequent apo-
ptosis (Muzio et al., 1998; Yang et al., 1998a). Similar
results were reported for the mammalian caspase-9
(Srinivasula et al., 1998a) and the C. elegans CED3 (Yang
et al., 1998b).
Although induced proximity undoubtedly leads to cas-
pase activation, it remains unclear whether this is indeed
how the initiator caspases are activated under physio-
logical conditions. For example, effector caspases can
also be autoactivated through induced proximity; yet
in vivo they are activated by the initiator caspases. In
addition, unprocessed procaspase-9 is nearly as active
as mature caspase-9 (Srinivasula et al., 2001) and the
primary function of the apoptosome is to allosterically
enhance caspase-9 activity rather than to facilitate its
autoactivation (Rodriguez and Lazebnik, 1999). Further-
more, forced oligomerization of the initiator caspases
may not recapitulate the physiological context, in terms
of protein expression levels, and more importantly, the
specific protein-protein interactions that are required
for the precise positioning and activation of the initiator
caspases.
Caspase-9 and the Apoptosome
It was a rather startling discovery that the processed
caspase-9 is only marginally active in the absence of the
apoptosome, prompting the concept of a holo-enzyme
(Rodriguez and Lazebnik, 1999). Through association
with the apoptosome, the catalytic activity of caspase-9
Molecular Cell
468
is enhanced more than 1000-fold (Rodriguez and Lazeb-
nik, 1999). Surprisingly, the unprocessed caspase-9 can
be similarly maintained in this “hyperactive” state, dem-
onstrating that the proteolytic processing is unneces-
sary for the activation of procaspase-9 (Srinivasula et
al., 2001).
The primary component of the apoptosome, Apaf-1,
comprises an N-terminal CARD domain, a CED4 homol-
ogy domain, and 13 repeats of WD40 at its C-terminal
half. The three-dimensional structure of the apoptosome
at 27 Å resolution reveals a wheel-shaped heptameric
complex, with the CARD domains of Apaf-1 located at
the central hub and the WD40 repeats at the extended
spokes (Acehan et al., 2002) (Figure 7A). Docking of
caspase-9 to this apoptosome resulted in a dome-
shaped structure in the center; however, the bulk of the
caspase-9 was not visible in these EM studies. Although
the concept of apoptosome had been biochemically es-
tablished, the visualization of the apoptosome allows
the identification of the relevant components and proves
the structural involvement of Cyt. c in this functional
assembly. This structure should also remove remaining
doubts about the integrity of the apoptosome as a single
structural entity. Nevertheless, because of the low reso-
lution, the identified domain organization remains to be
validated.
Although most other caspases exist exclusively as
homodimers in solution, under physiological conditions,
caspase-9 was found to exist in an equilibrium between
predominantly monomers and a small amount of dimers
(Renatus et al., 2001). Fractions corresponding to dimers
and monomers from gel filtration were separately ana-
lyzed for their catalytic activity, and only the dimer frac-
tions were found to be active. Interestingly, biochemical
as well as structural analyses revealed that dimerization
resulted in the formation of only one functional active
site (Renatus et al., 2001) (Figure 7B). Based on these
observations, it was proposed that dimer formation may
drive the activation of caspase-9. While these studies
provide solid leads on the mechanisms of caspase-9
activation, they also raise many questions. For example,
why didn’t the diluted dimers dissociate into the inactive
monomers after gel filtration? And why couldn’t the far
more concentrated monomers form active dimers? It
also remains to be seen how the extremely hydrophobic
dimerization interface is shielded from solvent in mono-
meric caspase-9.
Based on structural information on caspase-9 and
the apoptosome, a model was proposed to explain the
activation of procaspase-9 (Acehan et al., 2002) (Figure
7C). In this model, a heptameric apoptosome binds
seven monomers of inactive caspase-9. The high local
concentrations of caspase-9 within this apoptosome
drive the efficient recruitment of additional inactive cas-
pase-9 monomers, which become activated upon bind-
ing. This interesting model contains a strong assumption
that caspase-9 activity in the dimers is identical to that
in the apoptosome holoenzyme, which remains to be
experimentally examined.
Future Perspectives
Recent investigation on caspase regulation has led to
a significantly improved understanding of the activation
of procaspase zymogen, the mechanisms of IAP-medi-
ated caspase inhibition, and the Smac-mediated re-
moval of caspase inhibition by IAPs. The identification of
a conserved tetrapeptide IAP-binding motif helps unify
mechanisms of apoptosis regulation from fruit flies to
mammals. Despite these advances, many important
questions remain unanswered. Our understanding of
how the initiator caspases are activated is far from com-
plete. Although the functions of the BIR2 and BIR3 do-
mains are well understood in XIAP, c-IAP1 and c-IAP2,
it is unclear what role BIR1 plays in apoptosis and how
the different BIR domains cooperate in the full-length
IAP to regulate cell death. In addition, it remains to be
examined whether the principles derived from mamma-
lian caspases are generally applicable to other species
in which no structural information is yet available. Future
structural and biochemical studies should clarify these
issues. Furthermore, several of the known caspases
function primarily in the inflammatory response rather
than in programmed cell death. Thus, activation of a
caspase cascade does not always lead to apoptosis.
Elucidation of the pathways involving these caspases
will reveal important insights into how different cas-
pases regulate distinct biological processes.
Acknowledgments
I would like to thank members of my laboratory for discussion, C.
Akey for help with the preparation of Figure 7, and F. Hughson
for helpful comments. This research is supported by the National
Institutes of Health (R01-CA90269), the Searle Scholar Foundation,
and the Rita Allen Foundation.
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