Tài liệu Báo cáo khoa học: Plasticity of S2–S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis pdf

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.. However, a pept

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 into three groups based on their function and

Keywords

allosteric site; apoptosis; cysteine protease;

enzyme

Correspondence

I T Weber, Department of Biology, Georgia

State University, PO Box 4010, Atlanta,

GA 30302, USA

Fax: +1 404 413 5301

Tel: +1 404 413 5411

E-mail: iweber@gsu.edu

Database

The atomic coordinates and structure

factors have been deposited in the Protein

Data Bank under the accession codes 2QL5

for caspase-7 ⁄ DMQD, 2QL9 for caspase-7 ⁄

DQMD, 2QLF for caspase-7 ⁄ DNLD, 2QL7

for caspase-7 ⁄ IEPD, 2QLB for caspase-7 ⁄

ESMD, and 2QLJ for caspase-7 ⁄ WEHD

(Received 18 April 2007, revised 3 July

2007, accepted 17 July 2007)

doi:10.1111/j.1742-4658.2007.05994.x

Many protein substrates of caspases are cleaved at noncanonical sites in comparison to the recognition motifs reported for the three caspase sub-groups To provide insight into the specificity and aid in the design of drugs to control cell death, crystal structures of caspase-7 were determined

in complexes with six peptide analogs (Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho, Ac-WEHD-Cho) that span the major recognition motifs of the three subgroups The crystal structures show that the S2 pocket of caspase-7 can accommodate diverse 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.

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

for DEXD, and caspase-6, 8 and 9 prefer (L⁄ V)EXD

[21,22] However, a peptide library search with

sub-strate-phage identified DLVD with 170% faster

hydro-lysis by caspase-3 than the canonical DEVD peptide,

thereby challenging the specificity of group III

execu-tioner caspases [23] Similarly, screening of peptide

inhibitors based on amino acid positional fitness scores

predicted DFPD as a potent inhibitor of caspase-7

[24] Also, the DNLD peptide was shown to have

simi-lar potent inhibitory activity on caspase-3 as the

canonical DEVD [24] The crystal structure of

caspase-8 in complex with the assumed non-optimal inhibitor

Ac-DEVD-Cho questioned the original specificity

clas-sification [25] Moreover, the specificity extends beyond

the P4–P1 tetrapeptide Our previous study has

identi-fied a preference of caspase-3 for hydrophobic P5

resi-dues, unlike caspase-7 [26], but similar to caspase-2

[27] Recently, an increasing number of caspase

sub-strates have been found to be cleaved at noncanonical

sites [28] These studies undermine the current

classifi-cation of caspase specificity into three groups

There-fore, new structural data are needed to determine the

detailed interactions that define caspase specificity

The structure of caspase-7 in complex with the

canonical tetrapeptide inhibitor DEVD is known [29]

In further studies of the molecular basis for the

sub-strate specificity of executioner caspases, we have

determined the crystal structures of human

recombi-nant caspase-7 in complexes with six tetrapeptide

analogs: Ac-DMQD-Cho, Ac-DQMD-Cho,

Ac-DNLD-Cho, Ac-IEPD-Ac-DNLD-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-Ac-DMQD-Cho, Ac-DNLD-Ac-DMQD-Cho, Ac-IEPD-Ac-DMQD-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 and caspase-8, whereas WEHD forms the optimal sub-strate sequence of inflammatory caspases [22] The kinetic parameters of caspase-7 were measured for the canonical substrate Ac-DEVD-pNA The Kmvalue for this substrate is 54.5 ± 2.18 lm whereas the kcat⁄ Kmis 4071.6 mm)1Æmin)1 The inhibitory potency of the six aldehydes together with the canonical Ac-DEVD-Cho can be divided into two groups based on the Kivalues The first group consists of stronger inhibitors with rela-tively low Ki values in the order of Ac-DEVD-Cho (0.7 ± 0.03 nm) Ac-DQMD-Cho (0.94 ± 0.04 nm)

< Ac-DNLD-Cho (1.4 ± 0.06 nm) << Ac-DMQD-Cho (8.0 ± 0.3 nm) The second group contains weaker inhibitors with much higher Ki values in the order of Ac-IEPD-Cho (550 ± 22 nm) < Ac-ESMD-Cho (1300

± 50 nm) << 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, DQMD-Cho, DNLD-Cho, IEPD-Cho, Ac-ESMD-Cho and Ac-WEHD-Cho All the complexes crystallized in the trigonal space group of P3221 (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

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

heterodimers were even more similar to those of

cas-pase-7⁄ DEVD complex showing rmsd of 0.24–0.33 A˚

and 0.24–0.35 A˚ for 230 equivalent Ca atoms The

conformations of the four prominent surface loops

that form the substrate-binding cleft of caspase-7 agree

well with those of caspase-7⁄ DEVD complex

There-fore, the substrate analogs of diverse sequences can be

accommodated in the substrate binding cleft without

major changes in overall conformation

Conformation of peptide analogs

The peptide analog inhibitors of the six complexes were

clearly visible in the electron density maps The electron

density for the inhibitor in the caspase-7⁄ DQMD

struc-ture is shown in Fig 1B The peptide inhibitors adopt

an extended conformation in all the complexes The inhibitors bind with thiohemiacetal bonds between the aldehyde group (-CHO) and the mercapto group (-SH)

of Cys186 of caspase-7 The peptide inhibitors make most of their contacts with the residues from the p10 subunit, except for those with the catalytic dyad (Cys186 and His144) and the residues anchoring the aspartate residue in the P1 position (Arg87, Ala145 and Gln184) which are in the p20 chain The inhibitors bind the two active sites in the heterotetramer in a similar fashion except for the alternate conformations for P3 Ser and P4 Trp in ESMD and WEHD complexes, which are described below The Ca positions of the 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

Refinement statistics

Number of atoms

Citrate ion

rmsd

a Values in parentheses are given for the highest resolution shell b Rsym¼ S hkl |Ihkl) ÆI hkl æ| ⁄ S hkl Ihkl c R ¼ S|F obs ) F cal | ⁄ SF obs

d Rfree¼ S test (|Fobs| ) |F cal |) 2 ⁄ S test |Fobs| 2

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

Cy s 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

subunits and their symmetry related equivalents p20¢ and p10¢ are

shown in green, blue, red and cyan, respectively The catalytic

cysteines in the p20 subunits are shown in green and red ball and

stick The magenta ball and stick model represents the

Ac-DQMD-CHO inhibitor (B) 2Fo-Fcelectron density map of Ac-DQMD-CHO in

the caspase-7⁄ DQMD structure contoured at a level of 1r The

cat-alytic Cys186 of caspase-7 forms a thiohemiacetal bond with the

acetyl group of the inhibitor.

Cys 186

A

B

P1

P2

P3

P4

Tyr 230

Arg 87

Pro 235

Arg 233

Trp 232 Phe 282

Gln 276 Trp 240

Cys 186

P1 Arg 87 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.

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 pocket and must rotate approximately 90 to accom-modate the P2 residue [33] Similarly, the S2 pocket

Fig 3 Schematic diagram of caspase-7 interactions with peptide analog inhibitors (A) Caspase-7 ⁄ DQMD (B) Caspase-7 ⁄ DNLD (C) Cas-pase-7 ⁄ DMQD (D) Caspase-7 ⁄ IEPD (E) Caspase-7 ⁄ ESMD (F) Caspase-7 ⁄ WEHD Thicker lines represent the peptide analog inhibitors The inhibitors are covalently bound to catalytic Cys186 Dashed lines represent hydrogen bonds and salt bridges, while curved lines indicate van der Waals interactions.

was blocked in the crystal structure of caspase-3 in

complex with the inhibitor of apoptosis protein XIAP

[34] The S2 subsites of caspase-7 and 3 are formed by

identical aromatic residues (Tyr230, Trp232 and

Phe282 in caspase-7) The inflammatory and initiator

caspases have larger S2 subsites due to the substitution

of Tyr230 with Val and tolerate bulkier side chains

The S2 subsites in caspase-3 and 7 were predicted to

preferentially accommodate small b-branched aliphatic

residues such as Val and Thr29

To investigate the plasticity of the S2 pocket of

caspase-7, it was probed with P2 Gln, Met, Pro, Leu

and His in the six complexes with different

tetra-peptides (Figs 2 and 3) The complexes of caspase-7

with DNLD, DQMD, and ESMD contain the long

aliphatic Leu and Met at P2 The S2 subsite

accommo-dates Met and Leu with good hydrophobic interactions

by the rotation of the Phe282 and Tyr230 side chains to

expand the pocket (Fig 2 and supplementary Fig S1)

The Met side chains exhibit different conformations in

the complexes with DQMD and ESMD as shown in

supplementary Fig S1 Caspase-7⁄ DMQD contains

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 v2 angle of Tyr230 rotates more than 70 to form an aromatic stacking interac-tion with the P2 His This stacking interacinterac-tion is fur-ther strengthened by the hydrogen bond between NE2

of P2 His and OH of Tyr230 in one binding site These structures demonstrate that the size of the S2 pocket

of caspase-7 can be enlarged or reduced by rotating

Tyr 230

A

D

B

E

C

F

Phe 282

Trp 232

Trp 232 Phe 282

Tyr 240

P2 Pro

P2 His

Arg 233

P3 Ser Pro 235

Val 86

Gln 276

Trp 232 Trp 232 Trp 240

Trp 240

P4 Glu

Gln 276

P4 Ile

Trp 240

Trp 420

P4 Ile

P4 Ile

P3 Glu

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.

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

bonds stabilize and fix the P3 residue in position,

inde-pendent of the type of side chain The side chain of

the canonical glutamic acid in the caspase-7⁄ IEPD and

caspase-7⁄ WEHD complexes forms favorable ionic

interactions with the guanidinium group of Arg233

(supplementary Fig S2) The long polar P3 residues

Gln and Asn in complexes with DQMD and DNLD

form similar hydrogen bond interactions with Arg233

However, the hydrophobic side chain of P3 Met in the

complex with DMQD exhibits different conformations

in the two active sites In one conformation, it is

direc-ted into the subsite whereas, in the second active site,

the Met side chain is directed out of the S3 groove

and has hydrophobic interactions with Pro235 The

CB and CG atoms of Met P3 are positioned similarly

to the equivalent atoms of P3 Glu The smaller polar

P3 Ser in the caspase-7⁄ ESMD complex is positioned

to form a hydrogen bond interaction with Arg233 in

one binding site, in addition to water-mediated

interac-tions with both Arg233 and Val86 (Fig 4C) In

sum-mary, the P3 residue is anchored by the main chain

hydrogen bonds with Arg233, and key interactions

necessary for the specificity of the S3 subsite are

con-served for both smaller and longer polar P3 residues

Moreover, hydrophobic P3 residues can form

favor-able van der Waals interactions with Pro235

S4 subsite

The structural divergence at the S4 subsite provides

major specificity conferring elements to the three

groups of caspases The S4 subsite of inflammatory

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 with the formation of a hydrogen bond with the main chain amide of Gln276 (Fig 4D) The P4 Glu also forms a hydrogen bond with the NE1 of Trp240 P4 Trp in the caspase-7⁄ WEHD complex was accom-modated in the S4 subsite by rotation of the side chains of Trp232 and Ser234 in addition to 1.2–1.6 A˚ shifts of residues 278–281 The P4 Trp exhibits differ-ent oridiffer-entations in the two binding sites However, there is no hydrogen bond between the P4 amide and carbonyl oxygen of Gln276 in both orientations Therefore, the structural changes confirm that large aromatic residues are not favored at the S4 subsite of caspase-7 (supplementary Fig S3) The Ile P4 in the caspase-7⁄ IEPD complex fits snugly between Trp232 and Trp240 and forms favorable van der Waals inter-actions with Trp232, Trp240 and CB of Ser275 (Fig 4E) Thus, Trp232 plays a dual role in caspase-7

by interacting with both P2 and small, branched ali-phatic P4 residues Interestingly, a similar hydrophobic S4 subsite and mode of binding of P4 Ile was observed with the initiator caspase-8 where the P4 Ile side chain lies between Trp420 and Tyr412 (Fig 4F) This struc-tural analysis implies that the S4 subsite of caspase-7

is well suited for Glu as well as Asp at P4 Furthermore,

small aliphatic residues are well tolerated in the S4

pocket and form favorable hydrophobic interactions

with Trp232 and Trp240 However, the large aromatic

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-Ac-DNLD-Cho) and weak (Ac-IEPD-Ac-DNLD-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 Ki 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-ESMD-cho is a weaker inhibitor than those with P4

Asp The polar interaction of Arg233 with the P3 Ser

side chain hydroxyl of Ac-ESMD-cho is lost in one

binding site Also, the P2 Met in one of the binding sites

of caspase-7⁄ ESMD complex is moved out relative to

the position of the P2 Met side chain in the

caspase-7⁄ DQMD complex These changes at the S2 and S3

subsites reduce the overall inhibitory potential of

Ac-ESMD-cho compared to P4 Asp containing

inhibi-tors The weakest inhibitor is Ac-WEHD-Cho, which

agrees well with the larger structural changes in S4 when

the bulky tryptophan residue is at the P4 position

Moreover, unlike the inhibitors with P4 Asp, there are

no hydrogen bond interactions of caspase-7 with P4 Trp

in the caspase-7⁄ WEHD structure (Fig 3)

Bound citrate at the allosteric site of caspase-7

A citrate molecule was found at the allosteric

inhibi-tory site at the dimer interface of caspase-7 The

compounds DICA and FICA can bind covalently to Cys290 resulting in movement of Tyr233 and the cata-lytic Cys186 away from the active conformation, which prevented binding of tetrapeptide inhibitors at the active site [36] A similar allosteric site and inhibitory mechanism were observed in inflammatory caspase-1 [37] In five of our caspase-7 complexes, a citrate mole-cule, presumably an artifact from the crystallization solution, was observed at the allosteric site (Fig 5A)

O4 and O6 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 O1of 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 binding, the sequence could not be identified without ambiguity The two glycine residues in the sequence cannot be distinguished in the electron density from larger amino acids with disordered side chains The peptide is presumed to be part of a bacterial protein trapped from cell lysate during purification of caspase-7 The pentapeptide buries approximately 270 A˚2 of accessible surface area, mostly from the p10 subunit The central His is buried in a deep cavity, which is equidistant at approximately 30 A˚ from the two cata-lytic cysteines (Fig 5E) This deep cavity is formed by the residues Glu257, Gln260, Glu298 and Tyr300 from both p10 subunits (Fig 5D), whereas Gln59 from both p20 subunits flanks the surface of the cavity The His side chain interacts with the side chains of Glu298 of one p10 subunit and Gln260 of the other p10 subunit

It forms water-mediated interactions with Glu298 and

Tyr300 of the first p10 subunit and with Glu257 from

both p10 subunits The N-terminal amide of the

penta-peptide interacts with the side chain of Gln59 from the

p20 subunits The numerous interactions observed for

the bound peptide suggest that this cavity may have a

functional role

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-lenges the specificity requirements suggested by in vitro studies with short synthetic peptides These six new structures have demonstrated that the S2, S3 and S4 specificity subsites of executioner caspase-7 are more flexible than anticipated The enzyme accommodates noncanonical tetrapeptides in a similar manner as the

Cys 290 Tyr 233

Tyr 233’

Arg 187’

Cys 290’

C

CIT

O 6

O 5

O 2

O 1

O 3

O 4 O 7

~ 30 Å

~ 30 Å

Ac tive

site

Active site

Arg 187

Tyr 300’

Glu 257 Glu 257’

Glu 298

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) 2Fo-Fcelectron 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 electroelectro-static potential, red depicts areas of negative electroelectro-static potential and white represents areas of neutral potential The putative exosite is highly electronegative and equidistant from the two active sites.

canonical DEVD without dramatic conformational

changes The S2 subsite of caspases was suggested to

contribute to substrate recognition [17,42] The

aro-matic S2 subsite of caspase-3 and 7 containing bulky

Tyr230, Trp232 and Phe282 was predicted to

preferen-tially accommodate small aliphatic P2 residues such as

Ala, Val or Thr By contrast, the substitution of

Tyr230 by Val in inflammatory and initiator caspases

was suggested to account for the accommodation of

large P2 side chains Furthermore, screening of peptide

inhibitors based on amino acid positional fitness score

predicted that bulky polar His was the least favorable

residue at P2 for caspase-7 [24] However, our crystal

structures show that executioner caspases can

accom-modate both smaller and larger P2 residues with

favor-able interactions in the S2 subsite The rotation of the

side chains of Tyr230, Trp232 and Phe282 can reduce

or expand the S2 subsite of caspase-7 to accommodate

various sizes of hydrophobic P2 residue The aromatic

side chain of P2 His forms stacking interactions and a

hydrogen bond with Tyr230, which suggests that a

P2 Tyr is likely to bind in a similar orientation Several

physiological substrates of executioner caspase have

His or Tyr at P2 (supplementary Table S2) supporting

the structural observation; a more detailed list of

cas-pase substrates is provided elsewhere [28,32] In

addi-tion, inhibition data show that Ac-DMQD-Cho,

Ac-DQMD-Cho, Ac-DNLQ-Cho are as potent as the

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-dily accommodate P4 Trp By contrast, executioner caspases showed a absolute specificity for Asp at the P4 position in studies with fluorescent peptide sub-strates [35] A combinatorial tetrapeptide study showed that executioner caspases prefer Asp at P4, whereas initiator caspases accommodate Leu⁄ Ile ⁄ Val This classification was challenged by the observation that caspase-8 tolerates small hydrophobic Ile and acidic Asp residues at the P4 position [25] Our results con-firm that P4 Trp can bind but results in unfavorable structural distortions of the S4 pocket of caspase-7 Importantly, we show that the S4 pocket of caspase-7 has polar and nonpolar regions, which bind polar resi-dues or short-branched aliphatic side chains in a man-ner similar to that of initiator caspase-8 Trp232 of caspase-7 plays a dual role by interacting with hydro-phobic residues at both P2 and P4 Interestingly, several physiological substrates of executioner caspases have short-branched aliphatic P4 residues (supplemen-tary Table S2) For example, DCC (deleted in colorec-tal cancer), a candidate tumor suppressor gene, is cleaved by caspase-3 at the noncanonical LSVD sequence with an aliphatic P4 residue, and the cleavage product is proapoptotic [44] P4 Glu is also observed

in several physiological substrates, in agreement with our structure and kinetic data for the tetrapeptide ESMD

The plasticity of the specificity subsites of execu-tioner caspases demonstrated here suggests that factors other than the P4–P1 sequence contribute to substrate 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