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

0.006 1.3

0.006 1.3

0.006 1.3

0.006 1.3

0.006 1.3

0.007 1.3

Bond length (A˚ ) Angles ((cid:2))

a Values in parentheses are given for the highest resolution shell. b Rsym ¼ Shkl|Ihkl ) ÆIhklæ| ⁄ ShklIhkl. c R ¼ S|Fobs ) Fcal| ⁄ SFobs. d Rfree ¼ Stest(|Fobs| ) |Fcal|)2 ⁄ Stest|Fobs|2.

for

stronger three the for 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

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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 those with the catalytic dyad subunit, except (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 for the alternate conformations for fashion except 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 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

J. Agniswamy et al.

Plasticity of caspase-7 specificity pockets

p10

A

Cys 186

A

Tyr 230

p20’

P1

Arg 87

P2

Arg 233

Phe 282

P3

Trp 232

Pro 235

Trp 240

p20

P4

p10’

Gln 276

Ace

B

P4 Asp

B

P3 Gln

Cys 186

Tyr 230

Arg 87

P1

P2

P2 Met

P1 Asp

P3

Phe 282

Trp 232

Cy s 186

Pro 235

Trp 240

P4

Gln 276

inhibitors bound at

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-Fc electron 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.

Fig. 2. Superposition of 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.

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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.

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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.

S2 subsite

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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(cid:2) to accom- modate the P2 residue [33]. Similarly, the S2 pocket

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Plasticity of caspase-7 specificity pockets

residues

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 (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.

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(cid:2) 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 structures demonstrate that the size of the S2 pocket of caspase-7 can be enlarged or reduced by rotating To investigate the plasticity of the S2 pocket of caspase-7, it was probed with P2 Gln, Met, Pro, Leu tetra- and His in the six complexes with different 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

Tyr 240

Tyr 230

A

B

C

Arg 233

Val 86

Phe 282

P2 His

Phe 282

P2 Pro

P3 Ser

Pro 235

Trp 232

Trp 232

D

E

F

Trp 240

Trp 232

Trp 232

Trp 232 Trp 412

Trp 240

Trp 240 Trp 420

P2 Pro P3 Thr

P4 Ile

P4 Ile P4 Ile

P4 Glu

P3 Glu P3 Glu

Gln 276

Gln 276

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.

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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

[35]. Caspase-8, which belongs substrates

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- to cent 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].

forms

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 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

specificity conferring elements

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The structural divergence at the S4 subsite provides to the three major groups of caspases. The S4 subsite of inflammatory caspases of group I is an extended, shallow hydropho- 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 orientations 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,

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tetrapeptide inhibitors at 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

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 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 O1 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

reduce the overall

The 6 substrate analog inhibitors can be divided into strong (Ac-DEVD-Cho (cid:2) 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 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 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

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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 A citrate molecule was found at the allosteric inhibi- the dimer interface of caspase-7. The tory site at

J. Agniswamy et al.

Plasticity of caspase-7 specificity pockets

A

B

C

Tyr 233

Cys 290’

Arg 187

O3

O5

Arg 187’

O6

O4 O7

Cys 290

CIT

O1

Tyr 233’

O2

Surface potential

>–<

Glu 257’

Glu 257

D

E

Gln 260

Glu 298’

Active site

Glu 298

Gln 260’

~ 30 Å

Tyr 300’

Gln 59

Gln 59’

Tyr 300

Ser 302’

~ 30 Å

Active site

Pentapeptide

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-Fc 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.

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.

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

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(PARP), which polymerase 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 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 the abundant nuclear enzyme been proposed for poly(ADP-ribose) is cleaved at DEVD-213flG by caspase-3 and 7 resulting

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Plasticity of caspase-7 specificity pockets

both caspase-3 and 7 show autoprocessing, which con- firms that P3 Ser is recognized by executioner caspases.

indicating that

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, the S2 pocket of caspase-7 can harbor longer P2 residues than the predicted small b-branched aliphatic Val and Thr. 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- is tal cancer), a candidate tumor suppressor gene, 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.

that suggest

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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 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, 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

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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 obtain the Km and Vmax values by fitting reaction velocities as described [46]. The catalytic constant kcat for Ac-DEVD- pNA was determined using the equation kcat ¼ Vmax ⁄ [E], where the enzyme concentration [E] was determined by active site titration during Ki determination, as described below.

each inhibitor were determined by

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 large subunits flank the pocket in executioner caspases and no role has been established for these short exten- sions. Thus, it is tempting to speculate that the N-ter- minal extensions of the procaspases might block the potential exosite and their removal starts the process of executioner caspase activation, as well as the expo- sure of the exosite. Clearly, further studies are required to identify the molecular basis for substrate recogni- tion of caspases.

Experimental procedures

The peptide analogs Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho and Ac- WEHD-Cho form a covalent bond between the aldehyde group (Cho) and the mecapto (-SH) group of Cys186 in caspase-7. The aldehyde inhibitors of caspases are classified as reversible inhibitors. For the measurement of inhibition constant Ki, caspase-7 was preincubated with the peptide analogs in assay buffer at room temperature for 15 min. After the addition of substrate, the reaction velocity was measured based on substrate hydrolysis. The inhibition constants of a dose-dependent curve described by Ki ¼ (IC50 ) 0.5[E]) ⁄ (1 + [S] ⁄ Km), where [E], [S] and IC50, respectively, corre- spond to active enzyme concentration, substrate concentra- tion and the inhibitor concentration needed for half maximum enzyme activity [47].

Expression and purification of caspase-7

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 (cid:2)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].

The recombinant human caspase-7 was expressed in Escher- ichia coli grown at 37 (cid:2)C in 5 L of induction media (20 gÆL)1 tryptone, 10 gÆL)1 yeast extract, 5 gÆL)1 NaCl, 0.4% glucose, 1 mm MgCl2, 0.1 mm CaCl2, pH 7.4) supple- mented with 100 mgÆL)1 of ampicillin. Expression of recombinant protein was induced by adding isopropyl thio- b-d-galactoside to an absorbance of 0.6 at 600 nm. Cells 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 (HisTrapTM 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.

Crystallization, X-ray data collection, structure determination and analysis

The crystal structures were solved by molecular replace- ment with the published structure of caspase-7 (1F1J) as the initial model using phaser [49]. The inhibitors were fitted into unambiguous electron density. The models were subjected to several rounds of refinement in cns [50] and model building with o [51]. Solvent molecules were inserted at stereochemically reasonable positions. The final refined models have good protein geometry with no disal- lowed / ⁄ w-values on the Ramachandran plots. Hydrogen bond interactions were identified by distances of 2.6 – 3.5 A˚ between hydrogen donor and acceptor atoms.

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

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and 7: key mediators of mitochondrial events of apopto- sis. Science 311, 847–851.

7 Nguyen JT & Wells JA (2003) Direct activation of the apoptosis machinery as a mechanism to target cancer cells. Proc Natl Acad Sci USA 100, 7533–7538.

8 Jia LT, Zhang LH, Yu CJ, Zhao J, Xu YM, Gui JH,

Surface area calculations were made with contact and areaimol [52]. The structures were superposed globally on the canonical caspase-7 ⁄ DEVD complex using 460 topologically equivalent Calpha atoms with lsqman of the Uppsala software factory. Molecular figures were prepared with molscript, raster3d [53], and pymol (http:// www.pymol.org).

Acknowledgements

Jin M, Ji ZL, Wen WH, Wang CJ, Chen SY, Yang AG (2003) Specific tumoricidal activity of a secreted proapoptotic protein consisting of HER2 antibody and constitutively active caspase-3. Cancer Res 63, 3257–3262.

9 Endres M, Namura S, Shimizu-Sasamata M, Waeber C, Zhang L, Gomez-Isla T, Hyman BT, Moskowitz MA (1998) Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J Cereb Blood Flow Metab 18, 238–247.

10 Wiessner C, Sauer D, Alaimo D & Allegrini PR, (2000) Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo. Cell Mol Biol (Noisy-le-Grand) 46, 53–62.

BF was supported in part by the Georgia State Uni- versity Research Program Enhancement award. ITW is a Georgia Cancer Coalition Distinguished Cancer Scholar. This research was supported in part by the Georgia State University Molecular Basis of Disease Program, the Georgia Research Alliance, and the Georgia Cancer Coalition. We thank the staff at the 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.

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Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corres- ponding author for the article.