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

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 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. 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 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-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 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 K m value for this substrate is 54.5 ± 2.18 lm whereas the k cat ⁄ K m is 4071.6 mm )1 Æmin )1 . The inhibitory potency of the six aldehydes together with the canonical Ac-DEVD-Cho can be divided into two groups based on the K i values. The first group consists of stronger inhibitors with rela- tively low K i 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 K i values in the order of 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 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 Protein databank code 2QL9 2QL7 2QLB 2QL5 2QLF 2QLJ Space group P3 2 21 P3 2 21 P3 2 21 P3 2 21 P3 2 21 P3 2 21 a ¼ b(A ˚ ) 87.16 87.94 88.25 87.26 87.46 88.50 c(A ˚ ) 187.42 187.55 188.29 187.71 185.83 187.22 b (°) 120 120 120 120 120 120 Resolution range 50–2.14 50–2.4 50–2.25 50–2.34 50–2.8 50–2.6 Total observations 295 498 90 499 126 436 136 298 63 618 86 151 Unique reflections 44 778 31 049 38 990 32 117 18 047 24 974 Completeness 97.6 (80.2) a 92.3 (59.7) 94.3 (68.3) 90.2 (52.0) 90.3 (91) 92.1 (60.7) <I ⁄ r(I)> 24.0 (3.2) 12.2 (2.2) 16.0 (2.6) 15.5 (3.0) 13.8 (2.6) 16.7(3.3) R sym (%) b 7.2 (31.9) 9.8 (39.2) 9.0 (34.3) 9.8 (34.2) 9.2 (43.0) 6.7 (34.8) 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 sym ¼ S hkl |I hkl ) ÆI hkl æ| ⁄S hkl I hkl . c R ¼ S|F obs ) F cal | ⁄SF obs . d R free ¼ S test (|F obs | ) |F cal |) 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 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) 2F o -F c 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. Cys 186A B P1 P2 P3 P4 Tyr 230 Arg 87 Pro 235 Arg 233 Trp 232 Phe 282 Gln 276 T rp 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. 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 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. Plasticity of caspase-7 specificity pockets J. Agniswamy et al. 4756 FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 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 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 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. 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 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 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, Plasticity of caspase-7 specificity pockets J. Agniswamy et al. 4758 FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 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-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- 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). O 4 and O 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 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 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 4759 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’ AB DE 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 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. Plasticity of caspase-7 specificity pockets J. Agniswamy et al. 4760 FEBS Journal 274 (2007) 4752–4765 ª 2007 The Authors Journal compilation ª 2007 FEBS 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 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 [...]... graphics Acta Crystallogr D Biol Crystallogr 50, 869–873 Plasticity of caspase-7 specificity pockets Supplementary material The following supplementary material is available online: Fig S1 S2 subsite of caspase-7 Fig S2 S3 subsite of caspase-7 Fig S3 S4 subsite of caspase-7 Table S1 Polar interactions of caspase-7 with peptide analogs Table S2 Examples of physiological caspase substrates with studied substitutions... 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) Plasticity of caspase-7 specificity pockets 7 8 Acknowledgements BF was supported in part by the Georgia... potential exosite and their removal starts the process of executioner caspase activation, as well as the exposure of the exosite Clearly, further studies are required to identify the molecular basis for substrate recognition of caspases Experimental procedures Expression and purification of caspase-7 The recombinant human caspase-7 was expressed in Escherichia coli grown at 37 °C in 5 L of induction media... 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, Of ce of Science, Of ce of Basic... Kodandapani L, Wu JC & Grutter MG (2000) Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase8-Z-DEVD-cho complex J Mol Biol 302, 9–16 26 Fang B, Boross PI, Tozser J & Weber IT (2006) Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition J Mol Biol 360, 654 – 666 27 Schweizer A, Briand C & Grutter MG, (2003) Crystal structure of. .. (ADP-ribose) polymerase during apoptosis Evidence for involvement of caspase-7 J Biol Chem 274, 28379–28384 42 Chereau D, Kodandapani L, Tomaselli KJ, Spada AP & Wu JC (2003) Structural and functional analysis of caspase active sites Biochemistry 42, 4151 – 4160 43 Denault JB & Salvesen GS (2003) Human caspase-7 activity and regulation by its N-terminal peptide J Biol Chem 278, 34042–34050 44 Mehlen... 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 caspase-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... from the two active sites and possibly 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 extensions Thus, it is tempting to speculate that the N-terminal extensions of the procaspases might block... determined by active site titration during Ki determination, as described below The peptide analogs Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho and AcWEHD-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. . .Plasticity of caspase-7 specificity pockets J Agniswamy et al modified with long branched poly(ADP-ribose) chain has a much higher affinity for caspase-7 compared to caspase-3, implying specific interactions between caspase-7 and ADP-ribose moiety However, no exosite has been identified in caspases so far The symmetric . Plasticity of S2–S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis Johnson Agniswamy, Bin Fang and Irene. 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