Plasticity of S2–S4 specificity pockets of executioner
caspase-7 revealed by structural and kinetic analysis
Johnson Agniswamy, Bin Fang and Irene T. Weber
Department of Biology, Molecular Basis of Disease, Georgia State University, Atlanta, GA, USA
Caspases, the key effector molecules in apoptosis, are
potential targets for pharmacological modulation of
cell death. Uncontrolled apoptosis due to enhanced
caspase activity occurs in nerve crush injury, stroke
and neurodegenerative diseases such as Alzheimer’s,
Parkinson’s and Huntington’s diseases [1–3]. On the
other hand, inadequate caspase activity is implicated in
cancer, autoimmune diseases and viral infections [4–6],
and a number of potential drugs are being developed
for selective induction of apoptosis in cancer cells [7,8].
The substrate-based peptide inhibitor zVAD-fmk pro-
vides substantial protection against stroke, myocardial
infarction, osteoarthritis, hepatic injury, sepsis, and
amyotrophic lateral sclerosis in animal models [9–11].
Small nonpeptide inhibitors are preferred for their
superior metabolic stability and cell permeability. Two
nonpeptide inhibitors are currently in phase II clinical
trials: IDN6556 for treatment of acute-tissue injury
disease and liver diseases [12], and VX-740 for treat-
ment of rheumatoid arthritis [13]. Knowledge of the
molecular basis for substrate specificity of caspases is
critical for design of therapeutic agents for selective
control of cell death.
Caspases are cysteine proteases that hydrolyze the
peptide bond after an aspartate residue [14–17]. Thir-
teen human caspases have been cloned and character-
ized to varying extents [18,19]. Caspases are classified

residues. Glu is not required at the P3 position because Ac-DMQD-Cho,
Ac-DQMD-Cho and Ac-DNLD-Cho with varied P3 residues are almost as
potent as the canonical Ac-DEVD-Cho. P4 Asp was present in the better
inhibitors of caspase-7. However, the S4 pocket of executioner caspase-7
has alternate regions for binding of small branched aliphatic or polar resi-
dues similar to those of initiator caspase-8. The observed plasticity of the
caspase subsites agrees very well with the reported cleavage of many pro-
teins at noncanonical sites. The results imply that factors other than the
P4–P1 sequence, such as exosites, contribute to the in vivo substrate speci-
ficity of caspases. The novel peptide binding site identified on the molecular
surface of the current structures is suggested to be an exosite of caspase-7.
These results should be considered in the design of selective small molecule
inhibitors of this pharmacologically important protease.
Abbreviation
PARP, poly(ADP-ribose) polymerase.
4752 FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS
prodomain structures. Caspases-1, 4, 5 and 11 have
roles in cytokine maturation and inflammatory
responses and consequently are called inflammatory
caspases (group I) [20]. The other family members are
involved in apoptosis. Caspases-2, 8, 9, 10 and 12
function upstream within the apoptotic signaling
pathways and are termed initiator caspases (group II).
Caspases-3, 6, 7 and 14 are activated by initiator
caspases and act as the immediate executioners of the
apoptotic process. These caspases are termed execu-
tioner or effector caspases (group III). The caspases
are reported to recognize tetrapeptide motifs in their
substrates. Caspases-1, 4 and 5 prefer the tetrapeptide
WEHD, whereas caspase-2, 3 and 7 have a preference

Cho, Ac-IEPD-Cho, Ac-ESMD-Cho and Ac-WEHD-
Cho. The sequences of these peptidyl inhibitors span
the range of recognition motifs reported for the three
groups of caspases. These new structures reveal that
non-optimal peptides for group III and optimal pep-
tides of group I and II can bind and form favorable
interactions within S2, S3 and S4 subsites of group III
caspase-7. Also, a new peptide binding site was identi-
fied for on the molecular surface distal to the active
site. The results demonstrate the plasticity of substrate
recognition by caspase-7, and will be valuable for the
design of inhibitors of this pharmacologically impor-
tant enzyme.
Results
Inhibition of caspase-7 by tetrapeptide aldehydes
Six substrate analog reversible inhibitors, Ac-DMQD-
Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho,
Ac-ESMD-Cho and Ac-WEHD-Cho, which span the
three functional and phylogenetic classes of caspase sub-
strates, were evaluated for inhibition of caspase activity.
The selection of tetrapeptide sequences used in the pres-
ent study was based on the known protein cleavage sites
of caspases. DMQD is the reported executioner caspase
cleavage site of protein kinase C delta, and caspase
cleavage at DQMD in baculovirus p35 transforms it to
a pancaspase inhibitor [30,31]. ESMD forms the N-ter-
minal cleavage site in caspase-3, whereas DNLD was
identified as a potent substrate for executioner caspases
by computational studies [24,32]. IEPD has been identi-
fied as the optimal cleavage sequence of granzyme B

Ac-IEPD-Cho (550 ± 22 nm) < Ac-ESMD-Cho (1300
±50nm)<< Ac-WEHD-Cho (4400 ± 175 nm).
Overall structure of the six caspase-7 complexes
Caspase-7 was crystallized in complex with six sub-
strate analog reversible inhibitors, Ac-DMQD-Cho,
Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-
ESMD-Cho and Ac-WEHD-Cho. All the complexes
crystallized in the trigonal space group of P3
2
21
(Table 1). The structures were refined to the resolu-
tions of 2.14–2.8 A
˚
and R-factors from 18.7–21.2%.
The overall structure of the six independently refined
complexes is essentially identical with a complete
catalytic unit of two p20–p10 heterodimers in the
J. Agniswamy et al. Plasticity of caspase-7 specificity pockets
FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 4753
asymmetric unit (Fig. 1A). The two heterodimers are
arranged side by side in the opposite orientation to
form a central 12 stranded b-sheet surrounded by 10 a
helices. The refined overall structures are very similar
to the reported structure of caspase-7 with the canoni-
cal inhibitor DEVD [29]. The complete catalytic unit
of two heterodimers can be superimposed with that of
caspase-7 ⁄ DEVD with rmsd of 0.28–0.38 A
˚
for 460
topologically equivalent Ca atoms. The individual

peptides from all the six complexes are very similar and
superimpose with rmsd of 0.08–0.41 A
˚
. The peptides
and the interacting caspase-7 residues have very similar
conformations for the three stronger inhibitors,
whereas more structural variation is observed for the
weaker inhibitors compared to the canonical caspase-
7 ⁄ DEVD structure (Fig. 2A,B). The main chain atoms
of the inhibitors in all the six complexes exhibit similar
hydrogen bond interactions, except for P4 N in cas-
pase-7 ⁄ WEHD that cannot interact with the carbonyl
of Gln276 (Fig. 3, supplementary Table S1). Caspases
are unique among proteases in their stringent specificity
Table 1. Crystallographic data collection and refinement statistics.
Caspase-7 ⁄
Ac-DQMD-Cho
Caspase-7 ⁄
Ac-IEPD-Cho
Caspase-7 ⁄
Ac-ESMD-Cho
Caspase-7 ⁄
Ac-DMQD-Cho
Caspase-7 ⁄
Ac-DNLD-Cho
Caspase-7 ⁄
Ac-WEHD-Cho
Protein databank code 2QL9 2QL7 2QLB 2QL5 2QLF 2QLJ
Space group P3
2

Refinement statistics
Resolution range 50–2.14 50–2.4 50–2.25 50–2.34 50–2.8 50–2.6
R
cryst
(%)
c
19.1 19.6 18.7 21.2 19.6 19.6
R
free
(%)
d
22.5 23.7 22.3 23.3 22.9 23.4
Mean B ) factor (A
˚
2
) 45.3 51.0 49.4 62.3 56.3 70.7
Number of atoms
Protein 3825 3823 3821 3828 3828 3853
Water 296 220 215 125 59 52
Citrate ion
rmsd
111101
Bond length (A
˚
) 0.006 0.006 0.006 0.006 0.006 0.007
Angles (°) 1.3 1.3 1.3 1.3 1.3 1.3
a
Values in parentheses are given for the highest resolution shell.
b
R

2
⁄S
test
|F
obs
|
2
.
Plasticity of caspase-7 specificity pockets J. Agniswamy et al.
4754 FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS
for P1 Asp. The side chain atoms of aspartate at P1
have very similar positions and superpose well.
However, the side chain positions of P2, P3 and P4 res-
idues of the peptides differ significantly in the com-
plexes. These differences will be discussed separately
for each subsite.
Ace
P4 Asp
P3 Gln
P2 Met
P1 Asp
Cys 186
p10
A
B
p20
p10’
p20’
Fig. 1. Structure of caspase-7 and peptide analog inhibitors. (A)
Ribbon diagram of caspase-7 tetrameric assembly. The p20, p10

P2
P3
Trp 232
Trp 240
Gln 276
Phe 282
Tyr 230
Pro 235
P4
Fig. 2. Superposition of inhibitors bound at the active site.
(A) Superposition of stronger inhibitors and surrounding caspase-7
residues. The inhibitor and active site residues in the caspase-
7 ⁄ DEVD complex (protein databank code: 1F1J) are colored by ele-
ment type whereas those of caspase-7 ⁄ DQMD, caspase-7 ⁄ DMQD
and caspase-7 ⁄ DNLD are colored blue, cyan and green, respec-
tively. The inhibitors are in ball and stick representation and the cas-
pase residues are shown in a stick model. (B) Superposition of
weaker inhibitors and active site residues. The caspase-7 ⁄ IEPD,
caspase-7 ⁄ ESMD, caspase-7 ⁄ WEHD complexes are colored red,
magenta and yellow, respectively. For sake of clarity, residues
His144 and Gln184 in S1 subsite are not shown.
J. Agniswamy et al. Plasticity of caspase-7 specificity pockets
FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 4755
S2 subsite
By contrast to the highly specific S1 subsite, the S2
pocket of caspases is the only subsite to show substan-
tial alteration upon substrate binding, which indicates
its importance in substrate recognition and regulation
of activity. In the unliganded structure of caspase-3,
the side chain of a tyrosine residue occupies the S2

the polar P2 Gln, which is a mismatch in the hydro-
phobic S2 pocket. However, the CB and CG atoms of
Gln form favorable van der Waals interactions with
residues in the subsite, similar to those of Met P2, with
minor changes in the S2 aromatic side chains (supple-
mentary Fig. S1). The polar atoms of Gln are directed
away from the pocket. The smaller hydrophobic
P2 Pro is present in the caspase-7 ⁄IEPD complex. The
Tyr230 side chain has a similar conformation to that
in the canonical caspase-7 ⁄DEVD structure, but the
side chains of Trp232 and Phe282 adjust to form
favorable van der Waals interactions with P2 Pro
(Fig. 4A). The S2 pockets of caspase-7 and 3 were pre-
dicted not to accommodate aromatic residues. A com-
putational study on amino acid preference at different
subsites of caspase-7 based on positional fitness scores
predicted His as the amino acid with the least score
for binding in the S2 subsite [24]. However, the P2 His
in the caspase-7 ⁄ WEHD complex can clearly be
accommodated in the S2 subsite, although there are
relatively large movements of the three aromatic side
chains forming S2 (Fig. 4B). The CB of histidine is in
a similar position as the CB of valine in the caspase-
7 ⁄ DEVD complex. The v
2
angle of Tyr230 rotates
more than 70° to form an aromatic stacking interac-
tion with the P2 His. This stacking interaction is fur-
ther strengthened by the hydrogen bond between NE2
of P2 His and OH of Tyr230 in one binding site. These

P3 Glu
Trp 232
Trp 412
P2 Pro
P3 Thr
Fig. 4. Key variations in S2, S3 and S4 subsites of caspase-7. (A) Pro in S2 subsite of casepase-7 ⁄ IEPD. The new structure is colored by ele-
ment type and caspase-7 ⁄ DEVD is shown in cyan. (B) His in the S2 subsite of caspase-7 ⁄ WEHD. Dashed lines represent the hydrogen bond
and ion pair interactions. (C) Ser in the S3 subsite of caspase-7 ⁄ ESMD. (D) Glu in the S4 subsite caspase-7 ⁄ ESMD and (E) Ile in the S4 sub-
site of caspase-7 ⁄ IEPD. (F) Comparison of P4 Ile in the S4 subsites of caspase-7 and caspase-8 ⁄ IETD. The caspase-7 residues are colored
by atom type, whereas those of caspase-8 are shown in green.
J. Agniswamy et al. Plasticity of caspase-7 specificity pockets
FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 4757
the v angles of Tyr230, Trp232 and Phe282. This
adjustment enables caspase-7 to accommodate smaller
or larger aliphatic as well as long polar or aromatic
residues in the S2 subsite.
S3 subsite
Glutamic acid is considered to be the preferred P3
residue for all human caspases [21]. The P3 Glu is
anchored by multiple interactions with the conserved
Arg233 of caspase-7, which also is critical for binding
of P1 Asp. However, several physiological substrates
of caspase have been identified with different amino
acids at the P3 position (supplementary Table S2) [32].
Therefore, the specificity of the S3 subsite in caspase-7
was probed with Met, Gln, Asn, Glu and Ser in the
six complexes (Figs 2 and 3). The main chain amide
and carbonyl oxygen of the P3 residue form strong
hydrogen bonds with the main chain carbonyl and
amide of Arg233 in all structures. These hydrogen

caspases of group I is an extended, shallow hydropho-
bic depression suitable for the binding of a P4 Trp
residue. By contrast, the two bulky tryptophan resi-
dues lining the S4 subsite of group II and III apoptotic
caspases considerably reduce the size of the groove.
Experimental and theoretical studies have suggested
that caspase-3 and 7 have very high specificity for
aspartic acid at P4 [22,24]. Absolute specificity of Asp
over Glu at P4 was shown for caspase-7 using fluores-
cent substrates [35]. Caspase-8, which belongs to
group II, was shown to have high specificity for Leu at
the P4 position [22]. However, structural analysis sub-
sequently showed that caspase-8 tolerates both hydro-
phobic Ile and acidic Asp at P4 [25].
The specificity of the S4 subsite was probed with
Asp, Ile, Glu and Trp in the six caspase-7 structures,
and demonstrated greater flexibility in this subsite
compared to S2 and S3 (Figs 2 and 3). The main chain
amide of P4 residue forms a hydrogen bond with the
carbonyl oxygen of Gln276 in all the structures except
caspase-7 ⁄ WEHD. The side chain of P4 Asp binds cas-
pase-7 through interaction with the main chain amide
of Gln276. The reported interaction between the side
chain of Gln276 and P4 Asp in the caspase-7 ⁄DEVD
complex [29] is absent in all these complexes, even in
those with P4 Asp. A network of three ordered water
molecules deep in the subsite interacts with the side
chain of P4 Asp, suggesting that caspase-7 can accom-
modate residues larger than Asp at P4. The P4 Glu in
the caspase-7⁄ESMD complex extends into the subsite

Trp cannot be accommodated without structural
changes and loss of a hydrogen bond interaction.
Correlation of structural interactions and
inhibition of the peptides
The 6 substrate analog inhibitors can be divided into
strong (Ac-DEVD-Cho  Ac-DQMD-Cho < Ac-DNLD-
Cho << Ac-DMQD-Cho) and weak (Ac-IEPD-Cho <
Ac-ESMD-Cho << Ac-WEHD-Cho) inhibitors (Fig. 2).
The stronger inhibitors all contain Asp at P4. However,
the predicted specificity of caspase-7 for Glu at P3
position is not required because Ac-DMQD-Cho,
Ac-DQMD-Cho and Ac-DNLD-Cho with different P3
residues are almost as potent as the canonical Ac-
DEVD-Cho with P3 Glu. Similarly, the structural and
kinetic data show that S2 can accommodate longer P2
residues than the predicted small, b-branched Val and
Thr. The intermediate K
i
value of Ac-IEPD-Cho con-
firms the structural identification of the small aliphatic
binding region of the S4 subsite of caspase-7. The struc-
ture of caspase-7 ⁄IEPD shows only one hydrogen bond
between the main chain atoms of caspase-7 and P4, and
lacks the second hydrogen bond observed for the P4
Asp side chain in the four better inhibitors. The second
weakest inhibitor is Ac-ESMD-Cho, although the crys-
tal structure shows little change compared to the com-
plexes with more potent inhibitors. Although Glu is
structurally well suited at the P4 position due to an addi-
tional weak hydrogen bond compared to Asp, the Ac-

6
of the citrate molecule form close O S
interactions with Cys290 from the two p10 subunits.
The citrate oxygens have ionic interactions with the
side chain of Arg187 from both p10 subunits. Tyr233,
the third important residue at the allosteric site, inter-
acts with O
1
of citrate and a water molecule in the two
subunits, respectively. However, the active conforma-
tion was observed for the catalytic Cys186 and the
loops L1 to L4 forming the substrate binding site.
Thus, the citrate ion binds and forms favorable inter-
actions within the allosteric site despite the occupation
of the active site by tetrapeptide inhibitors.
Putative exosite in caspase-7
In all six caspase-7 structures, extended difference den-
sity was observed at a surface pocket between the two
p20–p10 heterodimers (Fig. 5B,C). This surface pocket
is approximately 22 A
˚
distant from the allosteric site
at the dimer interface where citrate is bound, and on
the opposite side of the molecular surface. The differ-
ence density was fit by a five-residue peptide in
extended conformation with the sequence of Gln-Gly-
His-Gly-Glu. The identity of the residues was deduced
from the shape of the electron density and the poten-
tial interactions with caspase-7 residues. However, due
to the resolution limit of the structure and surface

A similar cavity is also present in the structures of
inflammatory and initiator caspases. The size and
charge of the cavity varies among the caspases. In
caspase-3, substitution of Gln260 by His completely
closes the cavity, indicating either the absence of a
ligand binding site or a difference in the preferred
ligand. Exosites, which are binding sites distant from
the active site, often play an essential role in the sub-
strate recognition and processing by proteases [38].
Exosites have been proposed to explain the discrepan-
cies between in vivo protein cleavage sites and peptide
substrates preferred by in vitro studies of caspases
[16,17,39]. A role for an exosite on caspase-7 has
been proposed for the abundant nuclear enzyme
poly(ADP-ribose) polymerase (PARP), which is
cleaved at DEVD-213flG by caspase-3 and 7 resulting
in a form that cannot synthesize ADP-ribose poly-
mers in response to DNA damage [40]. Caspase-7
processes PARP modified with long branched poly
(ADP-ribose) chains much more efficiently than does
caspase-3, suggesting the presence of specific interac-
tions between poly(ADP-ribose) and caspase-7 [41].
The small peptide binding site identified in the cur-
rent structures is a putative exosite of caspase-7.
However, further studies will be needed to identify
the protein substrate for the exosite and possible
effect on caspase-7 activity.
Discussion
An increasing number of caspase substrates have been
shown to be cleaved at noncanonical sites, which chal-

~ 30 Å
Ac
tive
site
Active
site
Arg 187
Tyr 300’
Glu 257 Glu 257’
Glu 298
Glu 298’
Gln 260
Gln 260’
Gln 59
Pentapeptide
Tyr 300
Ser 302’
Gln 59’
Surface potential >–<
Fig. 5. Allosteric site and putative exosite of caspase-7. (A) Interaction of citrate ion bound at the allosteric site of caspase-7 ⁄ DMQD.
Despite occupation of the allosteric site, the catalytic residues are in the active conformation. (B) 2F
o
-F
c
electron density map of pentapep-
tide. (C) Peptide bound at the putative exosite on the surface of caspase-7. The caspase-7 surface is colored according to the element type.
The central histidine of the pentapeptide is buried deep in the cavity. (D) The interactions of the pentapeptide in the cavity. The water mole-
cules are represented as red balls. (E) The molecular surface of the caspase-7 around the putative exosite colored according to the electro-
static potential. Blue depicts areas of positive electrostatic potential, red depicts areas of negative electrostatic potential and white
represents areas of neutral potential. The putative exosite is highly electronegative and equidistant from the two active sites.

canonical Ac-DEVD-Cho, indicating that the S2
pocket of caspase-7 can harbor longer P2 residues than
the predicted small b-branched aliphatic Val and Thr.
Apart from the P1 residue, Glu P3 was optimal in
all three groups of caspases using the combinatorial
tetrapeptide substrate library search [22]. The long Glu
side chain is considered necessary to form ionic inter-
actions with the conserved Arg233. However, our
results suggest that the main chain interactions
between the P3 residue and Arg233 are more impor-
tant for proper positioning of the P3 residue. All six
inhibitors, irrespective of the P3 side chain, have con-
served main chain interactions and are positioned simi-
larly in the S3 subsite of caspase-7. Other polar
residues (Ser, Asn and Gln) form favorable hydrogen
bond interactions with the guanidinium group of
Arg233. The kinetic studies show that the presence of
Gln, Asn or Met at P3 does not alter the inhibitory
potency of the substrate analogs. In fact, the N-termi-
nal processing sites in procaspase-7 and procaspase-3
have the sequences DSVD and ESMD, respectively,
which implies that P3 Ser is physiologically acceptable
by initiator caspases. In some cells, caspase-3 was
shown to remove the N-terminal peptide of caspase-7
before the activation by granzyme B [43]. Moreover,
both caspase-3 and 7 show autoprocessing, which con-
firms that P3 Ser is recognized by executioner caspases.
The S4 pocket exhibits significant variability in both
substrate specificity and inhibitor selectivity among the
three groups of caspases. Inflammatory caspases rea-

specificity. Indeed, solvent exposed, partially ordered
regions of proteins with non-optimal sequences might
be processed by active caspases without the need of
high binding affinity. However, the large number of
substrates processed at noncanonical sites implies that
exosites may contribute to caspase recognition of their
substrates. For example, Bid, the pro-apoptotic Bcl2
family member, is cleaved by caspase-8 at an LQTD
motif in a flexible loop, but a second potential site
IGAD in the same loop is not processed. The second
site is certainly accessible because it is targeted by
granzyme B in the mitochondrial pathway of apoptosis
[45]. Thus, it is postulated that important exosite-medi-
ated interactions preferentially guide caspase to the
first site or conversely steer the caspase away from the
second site [17]. Similarly, the more efficient cleavage
of PARP by caspase-7 rather than caspase-3 also sug-
gests the existence of exosites [17]. In addition, PARP
J. Agniswamy et al. Plasticity of caspase-7 specificity pockets
FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 4761
modified with long branched poly(ADP-ribose) chain
has a much higher affinity for caspase-7 compared to
caspase-3, implying specific interactions between cas-
pase-7 and ADP-ribose moiety. However, no exosite
has been identified in caspases so far. The symmetric
pentapeptide binding pocket identified in the present
study is equidistant from the two active sites and pos-
sibly serves as an exosite for caspase-7. The size and
charge of the central cavity differs between caspase-3
and 7. Furthermore, the two extended N-termini of the

were harvested after 4.5 h and suspended in 200 mL of
25 mm Tris ⁄ HCl, 5 mm imidazole, 25 mm NaCl, 0.1% tri-
ton X-100, 0.1 mgÆmL
)1
lysozyme, pH 7.5. The cell lysate
obtained by centrifugation was loaded on to a nickel affin-
ity column (HisTrap
TM
HP, Amersham, NJ, USA). The
caspase-7 was eluted from the column using a 20 mm to
1 m imidazole gradient. The sample was dialyzed against
50 mm Tris, 100 mm NaCl, 20 mm imidazole, 10 mm
dithiothreitol, pH 7.5 to remove excess imidazole. The sam-
ple was further purified by size exclusion chromatography
on a Superdex-75 column (Amersham) with 50 mm Tris,
100 mm NaCl, 10 mm dithiothreitol, pH 7.5 as buffer. The
purity of the resulting sample was assessed by SDS ⁄ PAGE.
Enzyme kinetic assays
Enzymatic activity of caspase-7 was determined using the
colorimetric caspase-3 ⁄ 7 substrate Ac-DEVD-pNA (Bio-
mol, Plymouth Meeting, PA, USA), where Ac is the acetyl
group and pNA is p-nitroanilide. Caspase-7 was preincu-
bated in assay buffer (50 mm Hepes, 100 mm NaCl, 0.1%
Chaps, 10% glycerol, 1 mm EDTA and 10 mm dithiothrei-
tol, pH 7.5) at room temperature for 5 min prior to the
addition of substrate at different concentrations. p-Nitro-
anilide released by the substrate hydrolysis was measured at
a wavelength of 405 nm using a Polarstar Optima micro-
plate reader (BMG Labtechnologies, Durham, NC, USA).
sigmaplot 9.0 (SPSS Inc., Chicago, IL, USA) was used to

constants of each inhibitor were determined by a
dose-dependent curve described by K
i
¼ (IC
50
) 0.5[E]) ⁄
(1 + [S] ⁄ K
m
), where [E], [S] and IC
50
, respectively, corre-
spond to active enzyme concentration, substrate concentra-
tion and the inhibitor concentration needed for half
maximum enzyme activity [47].
Crystallization, X-ray data collection, structure
determination and analysis
Caspase-7 was incubated at room temperature with each of
the six inhibitors at a 1 : 20 molar ratio. Crystals of the cas-
pase-7 complexes were grown in hanging drops at room
temperature by mixing 1 lL of protein solution (6 mgÆmL
)1
of protein) and reservoir solution (12.6–14.5% poly(ethylene
glycol) 3350, 0.3 m diammonium hydrogen citrate, 10 mm
dithiothreitol). The crystals were frozen with cryoprotectant
of 18% poly(ethylene glycol) 3350, 0.3 m diammonium
hydrogen citrate and 21% glycerol. Diffraction data were
collected at 100 °K on beamline 22-ID (SER-CAT) at the
Advance Photon Source, Argonne National Laboratory
(Argonne, IL, USA). All data were integrated and scaled
with HKL2000 [48].

SER-CAT beamline at the Advanced Photon Source,
Argonne National Laboratory, for assistance during
X-ray data collection. Use of the Advanced Photon
Source was supported by the US Department of
Energy, Office of Science, Office of Basic Energy Sci-
ences, under Contract No. DE-AC02-06CH11357.
References
1 Tacconi S, Perri R, Balestrieri E, Grelli S, Bernardini S,
Annichiarico R, Mastino A, Caltagirone C, Macchi B
(2004) Increased caspase activation in peripheral blood
mononuclear cells of patients with Alzheimer’s disease.
Exp Neurol 190, 254 – 262.
2 Hartmann A, Troadec JD, Hunot S, Kikly K, Faucheux
BA, Mouatt-Prigent A, Ruberg M, Agid Y, Hirsch EC
(2001) Caspase-8 is an effector in apoptotic death of
dopaminergic neurons in Parkinson’s disease, but path-
way inhibition results in neuronal necrosis. J Neurosci
21, 2247–2255.
3 Hermel E, Gafni J, Propp SS, Leavitt BR, Wellington
CL, Young JE, Hackam AS, Logvinova AV, Peel AL,
Chen SF et al. (2004) Specific caspase interactions and
amplification are involved in selective neuronal vulnera-
bility in Huntington’s disease. Cell Death Differ 11,
424–438.
4 Vucic D, Stennicke HR, Pisabarro MT, Salvesen GS &
Dixit VM (2000) ML-IAP, a novel inhibitor of apopto-
sis that is preferentially expressed in human melanomas.
Curr Biol 10, 1359–1366.
5 Volkmann X, Cornberg M, Wedemeyer H, Lehner F,
Manns MP, Schulze-Osthoff K, Bantel H (2006)

matory cytokines. J Neurosci 20, 4398–4404.
12 Hoglen NC, Chen LS, Fisher CD, Hirakawa BP,
Groessl T & Contreras PC (2004) Characterization of
IDN-6556 (3-[2-(2-tert-butyl-phenylaminooxalyl)-
amino]-propionylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-
phenoxy)-pentanoic acid): a liver-targeted caspase
inhibitor. J Pharmacol Exp Ther 309, 634–640.
13 Leung-Toung R, Li W, Tam TF & Karimian K (2002)
Thiol-dependent enzymes and their inhibitors: a review.
Curr Med Chem 9, 979–1002.
14 Denault JB & Salvesen GS (2002) Caspases: keys in the
ignition of cell death. Chem Rev 102, 4489–4500.
15 Nicholson DW, (1999) Caspase structure, proteolytic
substrates, and function during apoptotic cell death.
Cell Death Differ 6, 1028–1042.
16 Thornberry NA & Lazebnik Y (1998) Caspases: enemies
within. Science 281, 1312–1316.
17 Fuentes-Prior P & Salvesen GS, (2004) The protein
structures that shape caspase activity, specificity, activa-
tion and inhibition. Biochem J 384, 201–232.
18 Degterev A, Boyce M & Yuan J (2003) A decade of
caspases. Oncogene 22, 8543–8567.
19 Lamkanfi M, Kalai M & Vandenabeele P (2004)
Caspase-12: an overview. Cell Death Differ 11, 365–368.
20 Martinon F, Holler N, Richard C & Tschopp J (2000)
Activation of a pro-apoptotic amplification loop
through inhibition of NF-kappaB-dependent survival
signals by caspase-mediated inactivation of RIP. FEBS
Lett 468, 134–136.
J. Agniswamy et al. Plasticity of caspase-7 specificity pockets

Many cuts to ruin: a comprehensive update of caspase
substrates. Cell Death Differ 10, 76 – 100.
29 Wei Y, Fox T, Chambers SP, Sintchak J, Coll JT, Golec
JM, Swenson L, Wilson KP, Charifson PS (2000) The
structures of caspases-1-3-7 and -8 reveal the basis for
substrate and inhibitor selectivity. Chem Biol 7, 423–432.
30 Bump NJ, Hackett M, Hugunin M, Seshagiri S, Brady
K, Chen P, Ferenz C, Franklin S, Ghayur T, Li P et al.
(1995) Inhibition of ICE family proteases by baculovirus
antiapoptotic protein p35. Science 269, 1885–1888.
31 Emoto Y, Manome Y, Meinhardt G, Kisaki H, Khar-
banda S, Robertson M, Ghayur T, Wong WW, Kamen
R, Weichselbaum R et al. (1995) Proteolytic activation
of protein kinase C delta by an ICE-like protease in
apoptotic cells. EMBO J 14, 6148–6156.
32 Earnshaw WC, Martins LM & Kaufmann SH (1999)
Mammalian caspases: structure, activation, substrates,
and functions during apoptosis. Annu Rev Biochem 68,
383–424.
33 Ni CZ, Li C, Wu JC, Spada AP & Ely KR (2003) Con-
formational restrictions in the active site of unliganded
human caspase-3. J Mol Recognit 16, 121–124.
34 Riedl SJ, Renatus M, Schwarzenbacher R, Zhou Q, Sun
C, Fesik SW, Liddington RC, Salvesen GS (2001) Struc-
tural basis for the inhibition of caspase-3 by XIAP. Cell
104, 791–800.
35 Stennicke HR, Renatus M, Meldal M & Salvesen GS
(2000) Internally quenched fluorescent peptide substrates
disclose the subsite preferences of human caspases 1, 3,
6, 7 and 8. Biochem J 350, 563–568.

product induces apoptosis by a mechanism requiring
receptor proteolysis. Nature 395, 801–804.
45 Barry M, Heibein JA, Pinkoski MJ, Lee SF,
Moyer RW, Green DR, Bleackley RC (2000)
Granzyme B short-circuits the need for caspase 8
activity during granule-mediated cytotoxic T-lympho-
cyte killing by directly cleaving Bid. Mol Cell Biol 20,
3781–3794.
46 Howard AD, Kostura MJ, Thornberry N, Ding GJ,
Limjuco G, Weidner J, Salley JP, Hogquist KA, Chap-
lin DD, Mumford RA et al. (1991) IL-1-converting
enzyme requires aspartic acid residues for processing of
the IL-1 beta precursor at two distinct sites and does
not cleave 31-kDa IL-1 alpha. J Immunol 147, 2964 –
2969.
47 Maibaum J & Rich DH (1988) Inhibition of porcine
pepsin by two substrate analogues containing statine.
The effect of histidine at the P2 subsite on the inhibition
of aspartic proteinases. J Med Chem 31, 625–629.
48 Otwinowski Z & Minor W (1997) Processing of X-ray
diffraction data collected in oscillation mode. Methods
Enzymol 276, 307–326.
Plasticity of caspase-7 specificity pockets J. Agniswamy et al.
4764 FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS
49 McCoy AJ, Grosse-Kunstleve RW, Storoni LC & Read
RJ (2005) Likelihood-enhanced fast translation func-
tions. Acta Crystallogr D Biol Crystallogr 61, 458–464.
50 Brunger AT, Adams PD, Clore GM, DeLano WL, Gros
P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges
M, Pannu NS et al. (1998) Crystallography and NMR

J. Agniswamy et al. Plasticity of caspase-7 specificity pockets
FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 4765