Stefan J. Riedl and Yigong Shi
Abstract | Caspases, which are the executioners of apoptosis, comprise two distinct classes, the initiators and the effectors. Although general structural features are shared between the initiator and the effector caspases, their activation, inhibition and release of inhibition are differentially regulated. Biochemical and structural studies have led to important advances in understanding the underlying molecular mechanisms of caspase regulation. This article reviews these latest advances and describes our present understanding of caspase regulation during apoptosis.
Apoptosis, or programmed cell death, is central to the development and homeostasis of metazoans1–5. Dysregulation of apoptosis leads to a variety of human pathologies including cancer, autoimmune diseases and neurodegenerative disorders6–10. Since the concept of apoptosis was established in 1972 (REF. 11), research efforts have led to the identification of hundreds of genes that control the initiation, execution and regulation of apoptosis in several species3. Compelling evidence shows that the mechanism of apoptosis is evolutionarily conserved. Caspases are the central components of the apoptotic response12,13. Caspases (which are so-named as they are cysteine proteases that cleave after an aspartate residue in their substrates14) are a conserved family of enzymes that irreversibly commit a cell to die. Although the first caspase, interleukin-1β-converting enzyme (ICE; also known as caspase-1), was identified in humans15,16, the critical involvement of caspases in apoptosis was discovered in the nematode worm Caenorhabditis elegans, in which the indispensable gene ced-3 (cell-death abnormality-3) was found to encode a cysteine protease that closely resembles the mammalian ICE17,18. Since then, at least 14 distinct mammalian caspases have been identified, of which there are 11 in humans12. Caspases and their homologues have been reported in species that range from the nematode to the dipteran Drosophila melanogaster 19 and the lepidopteran Spodoptera frugiperda 20, and even the yeast Saccharomyces cerevisiae 21. Over the past decade, many key events in caspase regulation have been documented at the molecular and cellular level3,22. The earlier focus on the genetic and cellbiological characterization of caspases has now been complemented by biochemical and structural investigations, giving rise to an unprecedented level of clarity in our understanding of caspase function12,23–25. In this review, we describe the molecular mechanisms of caspase regulation by focusing on the known biochemical and structural features of caspases and their regulators in organisms from fruitflies to humans.
Initiator and effector caspases

Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA. Correspondence to Y.S. e-mail:

Although the first mammalian caspase (caspase-1 or ICE) was identified as an important regulator of the inflammatory response15,16, at least 7 of the 14 known mammalian caspases have important roles in apoptosis12,26. The apoptotic caspases are generally divided into two classes: the initiator caspases, which include caspase-2, -8, -9 and -10 in mammals and Dronc and Dredd in fruitflies; and the effector caspases, which include caspases-3, -6 and -7 in mammals and Drice, Decay, Damm, Dcp1 and Strica in fruitflies (FIG. 1). CED-3 is the only apoptotic caspase in nematodes and functions as both an initiator and an effector caspase. An initiator caspase is characterized by an extended


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The p20 and p10 subunits closely associate with each other to form a caspase monomer. to its cell-surface DEATH RECEPTOR. AIF (apoptosis-inducing factor). fruitflies and nematodes. Once activated. a ~1. The effector and initiator caspases are shown in red and purple. which then cleaves and activates the effector caspase. which mediates the activation of caspase-9 (REFS 29. The intrinsic pathway is triggered in response to a wide range of death stimuli that are generated from within the cell. which binds to and activates the protein APAF1 in the cytoplasm29. forming an oligomeric death-inducing signalling complex (DISC)35. the removal of the prodomain is not required for the activation of caspases. Intrinsic and extrinsic pathways Fruitflies Drice 33 202 215 323 28 217 230 339 ~p20 Mammals Caspase-3 23 198 28 175 ~p10 277 303 Caspase-7 23 179 194 293 Effector caspases Caspase-6 216 374 385 479 Caspase-8 Caspase-10 Caspase-9 Caspase-2 DED DED DED 219 415 521 DED 315 331 416 Initiator caspases CARD 152 316 331 435 CARD L1 L2 L3 L4 Dcp1 Decay Damm Strica Dreddδ Dronc 323 Effector caspases 255 527 494 DED DED 144 324 351 450 Initiator caspases CARD L1 L2 L3 374 388 503 L4 Nematodes CED-3 CARD 220 L1 L2 L3 L4 Figure 1 | Apoptotic caspases in mammals. The activation of an effector caspase.REVIEWS Asp residues that separate the large (~p20) and small (~p10) subunits. respectively (FIG. so binding to their receptors leads to the formation of a minimally homotrimeric ligand–receptor complex that recruits further cytosolic factors. All caspases are produced in cells as catalytically inactive ZYMOGENS and must undergo proteolytic activation during apoptosis. Formation of the DISC leads to the activation of the initiator caspase. although the detailed molecular mechanisms remain unknown. The extrinsic pathway is initiated by the binding of an extracellular DEATH LIGAND. the activation of procaspase-9 is facilitated by the APOPTOSOME. removal of the N-terminal prodomain of a caspase is unnecessary for its catalytic activity. The p20 and p10 subunits together form a caspase monomer. The inactivation of this pathway is generally regarded as a hallmark of cancer7. see FIG. 2) — through cleavage at specific internal In mammalian cells. such as oncogene activation and DNA damage. the effector caspases are responsible for the proteolytic cleavage of a broad spectrum of cellular targets. Some of the well-characterized proteins include cytochrome c. 2). and. the presence of the prodomain is indispensable to the activation of initiator caspases. Unlike other protease zymogens. whereas an effector caspase usually contains 20–30 residues in its PRODOMAIN sequence.nature. PRODOMAIN The N-terminal amino-acid sequence of a caspase. The catalytic residue Cys is shown as a red line at the beginning of L2. For example. such as FADD and caspase-8. which comprises one or more adaptor domains that are important for its function. is carried out by an initiator caspase — such as caspase-9 and Dronc. N-terminal region. ~p20 and ~p10. Rather. By contrast. it is tightly regulated and often requires the assembly of a multi-component complex under apoptotic conditions27. As the activation of an initiator caspase in cells inevitably triggers a cascade of downstream caspase activation. The intrinsic pathway is mediated by mitochondria4. such as the caspase-recruitment domain (CARD) and the death-effector domain (DED). The extrinsic pathway can crosstalk to the intrinsic pathway through the caspase8-mediated cleavage of BID (a BH3-ONLY member of the 36.28. which then triggers the release of mitochondrial proteins. the apoptotic response is mediated through either the intrinsic pathway or the extrinsic pathway. respectively) is highlighted by a black arrow. EndoG (endonuclease G) and OMI/HTRA2 (high-temperature-requirement protein A2. the initiator caspases are auto-activated. The death ligands are constitutively homotrimeric. caspase-8. Perhaps the most intriguing one of these pro-apoptotic proteins is cytochrome c. several proteins are released from the intermembrane space of mitochondria into the cytoplasm4. The prodomains in initiator caspases invariably contain homotypic interaction motifs. For an overview of the 898 | NOVEMBER 2004 | VOLUME 5 ©2004 Nature Publishing Group www. SMAC (second mitochondria-derived activator of caspases)/DIABLO (direct inhibitor of apoptosis (IAP)-binding protein with low pI). such as Fas34. such as caspase-3 and Drice. depending on the origin of the death stimuli. caspase-3. The position of the first intra-chain activation cleavage (between the large and small subunits. which ultimately leads to cell death. These other cleavage events are thought to modulate caspase activity and the regulation of caspases by inhibitor of apoptosis (IAP) proteins and by other proteins. CED-3 (cell-death abnormality-3) is the only caspase in the nematode worm Caenorhabditis elegans and so fulfills the role of both initiator and effector caspase.4-MDa complex that includes APAF1 (apoptotic-protease-activating factor-1) and cytochrome c (see below)27. in response to apoptotic stimuli. The caspases and the location of functional segments are drawn to scale. thereby triggering a cascade of caspase activation.37 BCL2 FAMILY of proteins) .31–33). such as FasL. whereas other sites of cleavage are represented by grey arrows. Unlike other proteases. respectively. The four surface loops (L1–L4) that shape the catalytic groove are indicated. The function of the DISC in the activation of caspase-8 is thought to be analogous to that of the apoptosome in the activation of . The binding of cytochrome c to APAF1 induces a conformational change that allows APAF1 to bind to ATP/dATP and to form the apoptosome30.

and the C-terminal segment. EndoG. see the poster by J. The apoptosome recruits pro-caspase-9 and results in the allosteric activation of caspase-9. which is transcriptionally activated by cell-death stimuli. 44)) shares significant sequence similarity with the mammalian APAF1 and is important for the activation of the initiator caspase Dronc45 (FIG. which gives rise to a globular fold. Among the four loops. the negative regulation of CED-4 by CED-9 is removed by EGL-1.47). and forms in the presence of ATP or dATP. by an adaptor protein. During caspase activation. L1 and L3 have a relatively conserved length and composition among all the caspases. in turn. the BH3-only proteins (such as BID and BIM) transduce the signal to mitochondria after receiving apoptotic stimuli. form the base (FIG. 3b). whereas the L3 loop and the subsequent β-hairpin. 54) and Spodoptera frugiperda caspase-1 (REF. Cyt c. APAF1 and caspase-9 are the functional orthologues of the nematode CED-4 and CED-3 proteins. fruitflies and mammals (FIG. endonuclease G. which contains the catalytic cysteine. A conserved cascade of caspase activation The proteolytically inactive precursor of a protease. Hid. The inhibitor of apoptosis (IAP) proteins suppress apoptosis by negatively regulating the caspases. ced-9 and egl-1 — that function sequentially to control the onset of apoptosis1. both before and after the intra-chain activation cleavage. -8 (REFS 52. apoptotic-protease-activating factor-1. 2). 2). BH2 and BH3 domains) pro-apoptotic members of the BCL2 family of proteins 41. The basic structure of a caspase monomer is highly conserved. 3b). and are the functional homologues of. ced-4. APOPTOSOME A large protein complex that comprises cytochrome c and APAF1. NATURE REVIEWS | MOLECUL AR CELL BIOLOGY ©2004 Nature Publishing Group VOLUME 5 | NOVEMBER 2004 | 8 9 9 . mammals and fruitflies. with six β-strands from each monomer forming a single contiguous 12-stranded β-sheet (FIG. -2 (REF. whereas EGL-1 is a BH3-only member of the BCL2 family of proteins40.53) and -9 (REF. respectively (FIG. The release of mitochondrial proteins (such as cytochrome c) is mediated by BAK and BAX. 3a). structural information was restricted to an activated caspase that was bound to a covalent inhibitor12 (FIG. 3a). respectively (FIG. caspase-9 and caspase-3. apoptosisinducing factor. which stabilizes the active site of the neighbouring monomer. 2). Until a few years ago. CED-9 is a functional and structural homologue of the mammalian proteins BCL2 and BCL-XL39. Caspase-9 in mammals and Dronc in the fruitfly Drosophila melanogaster are initiator caspases. whereas caspase-3 and -7 in mammals and Drice in fruitflies belong to the class of effector caspases. Mechanism of effector-procaspase activation Caspases are synthesized as single-chain zymogens. 3a). Dronc. ZYMOGEN intrinsic and extrinsic apoptotic pathways. DEATH RECEPTOR The cell-surface receptor for the death ligand. high-temperature-requirement protein A2. whereas L2 and L4 are highly divergent. are located at two opposite ends of the β-sheet (FIG. During apoptosis. Several α-helices and short β-strands are located on either side of the central β-sheet. which involves the oligomerization of CED-4 (REF. -7 (REF. -7 Drice Cellular targets Apoptosis Cellular targets Apoptosis Figure 2 | A conserved apoptotic pathway in nematodes. which are formed by four protruding loops (L1–L4) from the scaffold. AIF. Such structures revealed that the backbone configuration of the active site in all caspases was highly conserved12 (FIG. Of the four active loops. 51). All isolated caspases were crystallized as homodimers (FIG. cleaves and activates the effector caspase Drice. Reed and Z. Huang entitled Apoptosis Pathways and Drug Targets in the online links box. 3a). L1 and L4 constitute two sides of the substrate-binding groove. CED-4 is constitutively suppressed by the anti-apoptotic protein CED-9 through direct physical interactions. Structural information is available for the mammalian caspase-1 (REFS 46.REVIEWS In mammals. DEATH LIGAND An extracellular growth factor that triggers an apoptotic response in cells. APAF1. In the absence of apoptotic signalling. HTRA2. BIM AIF HTRA2/OMI EndoG Reaper Hid Grim Sickle Fruitflies EGL-1 BCL2 CED-9 SMAC/DIABLO CED-4 Cyt c/dATP APAF1 Apaf1 Dronc Diap1 CED-3 Caspase-9 IAPs Apoptosis Caspase-3. the active-site conformation of one monomer is critically supported by the L2′ loop (the apostrophe denotes a different monomer) from the neighbouring monomer12 (FIG. whereas SMAC (second mitochondria-derived activator of caspases)/DIABLO (direct IAP-binding protein with low pI) in mammals and the RHG proteins Reaper. Genetic studies in nematodes have identified four genes — ced-3. CED-3 (cell-death abnormality-3) in the nematode worm Caenorhabditis elegans functions both as an initiator and effector caspase. 55). Homodimerization is mediated by hydrophobic interactions. In fruitflies. The caspase-activation pathway shows considerable similarity in nematodes. Grim and Sickle in fruitflies can remove the IAP-mediated negative regulation of caspases. containing the BH1. Dark 42 (also known as Dapaf-1 (REF. 43) or Hac-1 (REF. Dronc and Drice share considerable sequence similarity with. at least in part. the L2 loop is cleaved into the N-terminal segment. -3 (REFS 49. L2 harbours the catalytic cysteine and traverses the groove. Functional homologues of caspases and caspase regulators across species are indicated by the same colour. 48). 3). the ‘multidomain’ (that is. which are collectively referred to as L3. 38).50). 2). with a large (~20 kDa) and a small (~10 kDa) subunit. cytochrome c. Conserved structural features of caspases Nemodes Mammals Apoptotic stimuli Mitochondria BID. An effector caspase exists constitutively as a homodimer. The active sites. So. The activation of CED-3 is mediated. A death receptor contains an extracellular ligandbinding domain and an intracellular death domain. C. CED-4.

and the bound peptide inhibitor is shown in a ball-and-stick format. a | A schematic representation of procaspase-7 activation. This figure was prepared using the PyMol molecular graphics system (see the online links box). BH4). Although we do not yet understand how the initiator caspases are activated. As the interactions between L2′ and L2 and L4 are generally conserved12. the catalytic activity of an effector caspase is increased by several orders of magnitude after such cleavage. it is possible that steric hindrance contributes to the inactive conformation of the L3 loop and the lack of catalytic activity57. BH3. The BH3only members are pro-apoptotic. L2′ has an important role in stabilizing the conformation of the L2 and L4 loops. 3a). poised for catalysis. The intra-chain cleavage allows the L2′ and L2 loops to switch to their open conformation as observed in the inhibitor-bound caspase-7 (FIG. the intra-chain cleavage allows for the appropriate placement of the L3 loop. . In the active conformation. Note that L2′ stabilizes the active site of the adjacent caspase monomer. a | The structure of an inhibitor-bound caspase-7. Since this space is partly occupied by the intra-chain linker in the procaspase zymogen. 4a). These conformational rearrangements in the procaspase-7 zymogen prevent the formation of a substrate-binding groove. BH2. Strikingly. b | Structure of a procaspase-7 zymogen. is revealed by the conformational changes of the active site after the activation cleavage12 (FIG. BH2 and BH3. the mechanism of activation for a representative effector caspase. respectively. and the pro-apoptotic BH3-only protein family (such as BID. inverting the order of the large and small subunits at the primary-sequence level effectively frees the L2′ loop and is therefore predicted to activate caspases. so as to support the neighbouring active site. which contain BH1. In addition. the active-site loops undergo drastic conformational changes after cleavage. highlighted in red. the conformation of the active-site loops of the procaspase-7 zymogen does not support substrate binding or catalysis. However. Sequence alignment among the BCL2-family proteins has identified four BH domains. is positioned at one end of the groove. is flipped by nearly 180°. as well as for the fruitfly caspase Drice59. BAX and BAK). c | The L2′ loop has quite a different conformation in the procaspase-7 zymogen. this mechanism is likely to apply to other effector caspases. which provides critical support to the active site of the neighbouring caspase monomer. a Inhibitor L3 L4 L2′ L2 b L3 L1 L4 Cys L2′ L2 L4 L3 L1 Glu (P3) Asp (P1) Cys Asp (P4) Val (P2) L2′ L2 L1 L1′ L2′ L2 L3′ L4′ (from adjacent monomer) Figure 3 | Structural features of caspases. which contain four BH domains (BH1. compared with the inhibitor-bound active caspase-7. L2 is twisted. In this regard. thereby explaining why the procaspase-7 zymogen does not possess detectable catalytic activity. A diagram of the substrate-binding groove is shown in the upper left corner. and the four surface loops (L1–L4 and L1′–L4′) that constitute the catalytic groove of each caspase monomer are labelled and shown in green. The caspase core structure is shown in beige. 4c). 3a). L3 and L4 become highly flexible (FIG. proapoptotic multidomain proteins (for example. P1–P4 (shown in yellow) represent the four contiguous residues in the substrate that are recognized by the caspase. Unlike the inhibitor-bound active caspase-7. The L2′ loop. On the basis of this mechanism. is covalently bound to the peptide inhibitor. shifting the catalytic cysteine from its active conformation (FIG.nature. L1 and L4 constitute two parallel sides of the groove. caspase-7. the essence of effector-caspase activation is the ability of the L2′ loop to move freely. which locks the L2′ loop in the closed conformation and occludes it from stabilizing the active site. the unproductive conformation of the active site is a direct consequence of the uncleaved nature of the procaspase-7 zymogen. However. 58). Substrate or inhibitor Procaspase-7 zymogen Active caspase-7 (bound to substrate or inhibitor) L2′ L4′ L3′ L1′ Procaspase-7 zymogen Active caspase-7 (bound to inhibitor) Figure 4 | Molecular mechanism of procaspase-7 activation. The caspase core structure is shown in beige. L2. is shown. b | The active-site conformation of all known caspases is conserved. which harbours the catalytic residue Cys. BIM and PUMA). The active-site conformation before the activation cleavage is revealed by the structure of the unprocessed procaspase-7 zymogen56. The family is divided into antiapoptotic multidomain proteins (such as BCL2 and BCL-XL). In addition to the formation of the productive L2–L2′ interaction. The four surface loops (L1–L4 and L1′–L4′) that constitute the catalytic groove of each caspase monomer are labelled and shown in blue. whereas L3 serves as the base. the N-terminal region of L3 extends into the cavity between the two caspase monomers. 4c). BH1–BH4.REVIEWS remain unchanged. The active-site loops before and after the proteolytic processing are shown in green and blue. So. 4b). Compared with the inhibitor-bound active caspase-7 (FIG. the L2′ loop. 900 | NOVEMBER 2004 | VOLUME 5 ©2004 Nature Publishing Group www. BCL2 FAMILY a L2 L3 L4 L2′ Proteolytic processing L2′ L2 b L3 L1 L2 L4 L2′ c L3 L4 L2 L2′ L1 L2 L2′ A family of proteins that all contain at least one BCL2 homology (BH) region. this prediction was confirmed for mammalian caspase-3 and -6 (REF.57 (FIG. which is locked in a closed conformation by covalent linkage. is occluded from adopting its productive and open conformation. This figure was prepared using PyMol (see the online links box) and Molscript124. which is flexible in the zymogen. the core structural elements of the procaspase-7 zymogen BH3-ONLY BCL2 homology (BH) domain-3 only. which is representative of other caspase structures. The catalytic residue Cys.

respectively. The binding pockets for the P4-P3-P2-P1 positions in the substrate are known as the S4-S3-S2-S1 sub-sites. caspase-recruitment domain. which were originally identified in the genome of baculovirus on the basis of their ability to suppress apoptosis in infected host cells66. caspases-3. and cleave after the C-terminal residue (P1). There are eight mammalian IAPs. -7 Fruitflies Diap1 Diap2 Drice BIR1 BIR1 BIR2 438 BIR2 BIR3 RING RING 498 Figure 5 | IAPs in mammals and fruitflies. c-IAP2 and NAIP (REFS 65. The preferred P3 position is Glu for all caspases that have been examined12. which include Diap1 (also known as Thread) and Diap2 (FIG. In fruitflies. respectively. The P1 residue (Asp) is coordinated by three invariant residues at the S1 sub-site. as indicated in red. it was once thought that the substrate-binding grooves on caspases are pre-formed. which is usually an Asp residue12 (FIG. a Mammals Bruce/Apollon ILP2 ML-IAP/Livin Survivin NAIP c-IAP1 c-IAP2 XIAP BIR BIR1 BIR1 BIR1 BIR1 BIR BIR 142 1. Caspases are subject to transcriptional regulation and post-translational modifications26. On the basis of structural information. 63). The hallmark of IAPs is the baculoviral IAP repeat (BIR) domain. NAIP (neuronal apoptosis-inhibitory protein) and survivin. c-IAP1 or c-IAP2 (shown in red) is responsible for inhibiting caspases-3 and -7 in mammals. This mechanism might not be generally applicable to other caspases. an ~80-amino-acid zinc-binding domain70. Proteins of the inhibitor of apoptosis (IAP) family include XIAP (X-linked IAP). the third BIR domain (BIR3) potently inhibits the activity of processed caspase-9. Interestingly. which include XIAP (X-linked IAP). an Arg residue from L1. In XIAP. and are also known as MIHA/ILP1. Bruce/Apollon and survivin. the L2′ loop in the isolated active caspase-7 still exists in the closed conformation. The sequence specificity of caspase substrates is determined by the four active-site loops. although caspase-9 binds to several IAPs. c-IAP1. 3b). have also been found in mammals and fruitflies65. XIAP. c-IAP2. this caspase-3 was crystallized in the presence of excess inhibitors63.62. Because the backbone configuration of the active site for all inhibitor-bound caspases is highly conserved12. The Arg residue on L3 also coordinates the P3 residue (Glu).845 RING BIR2 BIR2 BIR2 BIR2 BIR3 604 BIR3 BIR3 BIR3 Caspase-9 Dronc CARD CARD RING RING 497 618 RING Caspase-3.403 BIR RING 236 4. in the structure of the free caspase-7. 56). A conserved linker peptide that precedes the BIR2 (baculoviral IAP repeat-2) domain of XIAP. ML-IAP (melanoma IAP)/Livin.69. the loops that form the substratebinding groove are flexible and quite different from those in the inhibitor-bound caspase-7 (REF. So. CARD. The IAP proteins.65.71. The preference in the P4 position varies among different groups of caspases and contributes to their substrate specificity. Surprisingly.REVIEWS Gln residue at the beginning of L2 and an Arg residue at the end of L3 (REF. but not in nematodes. respectively.5). by c-IAP1. but not Diap2. However. In mammals. ML-IAP (melanoma IAP)/Livin. 5). as recent structural evidence indicates that the active-site conformation of the isolated active caspase-3 closely resembles that of the inhibitorbound caspase-3 (REF. whereas the location of the S2 and S4 sub-sites is conserved12. P4-P3-P2-P1. 26). c-IAP1 and c-IAP2 contain three BIR domains each. The S1 and S3 sub-sites are nearly identical among all caspases. 5). are responsible for inhibiting the caspase-3 and -9 homologues Drice and Dronc. Only the BIR3 domain of XIAP can potently inhibit caspase9. and so the scenario of induced conformation cannot be ruled out. MIHC/HIAP1. Nonetheless. In fruitflies. it is primarily inhibited by XIAP.68).64. BIRC1 and TIAP. -7 and -9 are subject to inhibition by IAPs12 (FIGS 2. the BIR1 and BIR2 domains of Diap1. whereas the linker region between BIR1 and NATURE REVIEWS | MOLECUL AR CELL BIOLOGY ©2004 Nature Publishing Group VOLUME 5 | NOVEMBER 2004 | 9 0 1 . Dronc autoprocesses after a Glu residue60 and efficiently cleaves the fruitfly IAP Diap1 (see below) after a Glu residue61. MIHB/HIAP2. caspase-3 and -7 are inhibited by XIAP and. c-IAP1. binding of an inhibitor or substrate results in the observed conformational switch of the L2′ loop from a closed to an open form. ILP2 (IAP-like protein-2). to a lesser extent. The biochemically characterized BIR domains that have known functions are highlighted in colour. the P2 and P4 residues have greater sequence variation12. the conserved IAP family of proteins can potently inhibit the enzymatic activity of active caspases and can permanently remove caspases through the ubiquitylationmediated proteasome pathway12. mimicking the procaspase-7 zymogen and indicating that the isolated active caspase-7 adopts a conformation.67. Diap1 directly inhibits the catalytic activity of Drice and targets Dronc to the ubiquitylation-mediated degradation pathway61. ILP2 (IAP-like protein-2). c-IAP2. Ts-IAP. Structural studies using covalent peptide inhibitors have shown that these pockets are located mostly between the base (L3) and the two sides (L1 and L4) of the substrate-binding groove12. whereas other domains in the various IAPs are shown in grey. In addition. By contrast. residues 126–143 (FLLNKDVGNIAKYDIRVK) of NAIP are predicted to carry out this function. KIAP. NAIP (neuronal apoptosis-inhibitory protein). and the different BIR domains have distinct functions12 (FIG. and two fruitfly IAPs. Inhibitors of apoptosis 298 Mechanisms of substrate recognition Caspases recognize at least four contiguous amino acids in their substrates. The S2 and S4 sub-sites map mainly to the L3 and L4 loops. 56). which is intermediate between that of the zymogen and that of the inhibitor-bound caspase-7 (REF. Although caspases were thought to have an exclusive specificity for Asp.

Although the linker peptide that precedes the BIR2 domain of XIAP has an important role in inhibiting caspase-3 or -7. Asp148 of XIAP. 12). the linker peptide of XIAP occupies the active site of caspase-3 or -7 in a reverse orientation.REVIEWS BIR2 specifically targets caspase-3 and -7 (REF. the BIR2 domain of XIAP also makes direct interactions with caspase-3 (REF. So. the linker peptide fused to glutathione-S-transferase (GST) inhibited caspase-3 or -7 (REFS 77. Only processed caspase-9 is subject to inhibition by the BIR3 domain of XIAP. The structural information not only allows the identification of critical inhibiting residues in c-IAP1 and c-IAP2. the one defined by His343. These observations indicate a scenario in which the linker peptide needs to be presented in a competent conformation by a surrounding domain. 70). In contrast to the covalent peptide inhibitors. which occupies the S4 substrate-binding pocket. Mutation of His343 to Ala in XIAP did not affect binding to caspase-9 but did abolish inhibition 79. as the full-length XIAP shows a higher potency than the fusion protein between GST and the linker peptide77. forms hydrogen bonds with neighbouring residues in caspase-7. Another single-BIR-containing IAP. binding by XIAP is necessary but not sufficient for the inhibition of caspase-9. the BIR3 domain uses another surface patch. These mutational analyses further indicate two separate binding interfaces between caspase-9 and the BIR3 domain of XIAP. However. but also predicts that residues 126–143 of NAIP are responsible for inhibiting caspase-3 and -7 (FIG. 7a). an engineered protein with the linker peptide fused either N. Val146 makes a similar set of van der Waals contacts to surrounding caspase residues. 79). 7b). Two hydrophobic residues of XIAP. which prevents catalytic activity. does not inhibit caspase activity in vitro. In addition. make several van der Waals interactions with a conserved hydrophobic pocket on caspase-7. which was shown to be essential for the inhibition of caspase-3 (REF. Trp310 and Glu314 line the inner side of a conserved surface groove. ML-IAP/Livin. His343 is located on the opposite side of . although it does not seem to contain the sequence elements that are required for this inhibition. a | The crystal structure of caspase-7 (shown in beige and cyan) bound to an XIAP (X-linked inhibitor of apoptosis (IAP)) fragment (orange).81. which contains a single BIR domain. So. 6a). Val146 and Val147. this fragment is insufficient in isolation70. Compared with the covalent peptide inhibitors. binds the S4 pocket in a manner similar to the P4 residue (Asp) of the covalent peptide inhibitors. 902 | NOVEMBER 2004 | VOLUME 5 ©2004 Nature Publishing Group www. In the uninhibited state. Nonetheless. This interaction is essential to XIAP-mediated inhibition of caspase-9. 6b).77.nature. Mutational analysis identified three residues of XIAP — Trp310. The specific recognition of the caspase-9 dimerization interface requires four amino acids (including His343) in the BIR3 domain of XIAP. which are not a BIR2 C XIAP L3 L1 L4 N L2′ b Caspase-7 L2 XIAP C Asp148 Val146 L1′ Val147 Gly144 Caspase-7 L2′ L2 L4′ L3′ Figure 6 | Molecular mechanism of IAP-mediated inhibition of effector caspases. 76) and caspase-7 (REFS 77. 80) (FIG.or C-terminal to BIR1 was fully able to bind to and inhibit caspase-3. IAP-mediated inhibition of caspase-9. whereas neither BIR1 nor BIR2 showed any inhibition in isolation70. Asp148 of XIAP. 76). The molecular mechanism of IAP-mediated inhibition of effector caspases is revealed by the crystal structures of caspase-3 (REF. 7a).69. 80) (FIG. b | A close-up view of the active site of caspase-7 (in a surface representation) bound to an XIAP fragment (orange). These interactions are also conserved in the caspase-3–XIAP complex76. Survivin72. Mechanisms of caspase inhibition IAP-mediated inhibition of effector caspases. This trapped caspase-9 monomer adopts a catalytically incompetent conformation at the active site80 (FIG. 5). Glu314 and His343 — that are indispensable to XIAP-mediated inhibition of caspase-9 (REF.78). these three residues define two distinct surface areas in the BIR3 structure.78. as well as inhibited by XIAP. which has been shown to form the binding site of the N-terminal tetrapeptide of the small subunit of caspase-9. as does the P2 residue. This caspase-9 monomer has the potential to be activated by the apoptosome. the BIR domains also contribute to the inhibition of caspases. the second BIR domain (BIR2) is essential for the negative regulation of Dronc. which results in a blockade of substrate entry (FIG. Gly144 of XIAP is located close to the S1 pocket — within van der Waals contact distance of the catalytic cysteine in the caspases.78) bound to an inhibitory XIAP fragment. On the other hand. to heterodimerize with a caspase-9 monomer through the same interface that is required for the homodimerization of caspase-9 (REF. In support of this hypothesis. This figure was prepared using PyMol (see the online links box) and GRASP125. In these structures. XIAP inhibits caspase-9 by sequestering it in a monomeric state. However. The interaction between the conserved surface groove of the BIR3 domain of XIAP and the N-terminal tetrapeptide of the small subunit of caspase-9 anchors their mutual recognition80. a short linker peptide that precedes the BIR2 domain of XIAP forms identical interactions with caspase-3 or -7 (FIG. whereas BIR1 is responsible for inhibiting Drice61. The interactions primarily occur between a linker segment that is N-terminal to the BIR2 (baculoviral IAP repeat-2) domain of XIAP and the active site of caspase-7. Consistent with this observation. A mechanistic explanation for the inhibition of caspase-9 by XIAP was revealed by the crystal structure of caspase-9 bound to the BIR3 domain of XIAP (REF. In fruitfly Diap1. was reported to inhibit both caspase-3 and -9 (REFS 73–75). the processed caspase-9 is present exclusively as a monomer80.

Jafrac2. The catalytic residue. Serpin inactives protease by deformation of the active site. such as Met or Gly. followed by the subsequent formation of a covalently linked protease–inhibitor complex82–84.94). b | Superposition of the four active-site loops of the BIR3-bound caspase-9 (blue) and the active (yellow) and inactive (purple) monomers of the caspase-9 homodimer. respectively. SMAC/DIABLO and the RHG proteins has an essential function in the regulation of caspase function. and the zinc atom in the BIR3 domain of XIAP are shown in red. Caspase inhibition by p35 correlates with the cleavage of its reactive-site loop after residue Asp87. Another baculoviral protein. NATURE REVIEWS | MOLECUL AR CELL BIOLOGY ©2004 Nature Publishing Group VOLUME 5 | NOVEMBER 2004 | 9 0 3 . also contains an IAP-binding tetrapeptide motif at its N terminus (FIG. The crystal structure of caspase-8 in complex with p35 shows that the catalytic residue Cys360 of caspase-8 is covalently linked to Asp87 of p35 through a thioester bond85 (FIG. Caspase-9 and the BIR3 domain of XIAP are shown in beige and green. 9a). The next three residues of the tetrapeptide motif can tolerate some variation but strongly prefer Pro in the third position and a bulky. a | Structure of active caspase-9 bound to the BIR3 (baculoviral IAP repeat-3) domain of XIAP (X-linked inhibitor of apoptosis (IAP)). Hid. SERPIN A family of serine-protease inhibitors. In mammals. the SERPIN CrmA. In fruitflies. which calls into question whether HTRA2/OMI can antagonize XIAP inhibition under physiological settings. the baculoviral p35 protein potently inhibits most caspases. b | Close-up view of the covalent inhibition of caspase-8 by p35. a | Structure of caspase-8 (shown in blue and green) bound to the baculoviral caspase-inhibitor protein p35 (shown in orange and purple). functions similarly to p35 (REFS 86–88). with its methyl side chain fitting into a conserved hydrophobic pocket and its main-chain groups making hydrogen bonds to conserved surface residues in IAPs. contains a divergent tetrapeptide motif (FIG. In parallel. -7 and -9. Although a thioester bond is generally susceptible to hydrolysis. the IAP-mediated inhibition of caspases is antagonized by a family of proteins that contain an IAP-binding tetrapeptide motif 90 (FIG. 9a). Another mitochondrial protein. During apoptosis. RHG PROTEINS conserved in c-IAP1 or c-IAP2 (REF. A tetrapeptide IAP-binding motif a L1 L3 L2 L4 b L3 L3 BIR3 of XIAP L4 L2 L1 L2 Caspase-9 (bound to BIR3 of XIAP) Caspase-9 (inactive) Caspase-9 (active) Caspase-9 Figure 7 | Molecular mechanism of XIAP-mediated inhibition of caspase-9. Such recognition. 9a). p49. Named after the fruitfly Reaper. results in the abrogation of interactions with the BIR domains94. caspase-9. which specifically targets the mammalian caspases-3. which occludes access by water molecules85 (FIG. which is derived from the cowpox virus. but has nonetheless been shown to bind to Diap1 and to counter Diap1-mediated Dronc suppression102. The name now refers to a larger family of pro-apoptotic proteins in fruitflies that share an N-terminal inhibitor of apoptosis (IAP)-binding tetrapeptide motif. The BIR3 domain binds to a large caspase-9 surface that is normally required for its homodimerization. 9a). 8a). which are collectively referred to as the RHG PROTEINS. Grim and Sickle.103). the IAP-binding tetrapeptide motif that is found in caspase-9. Mutation of Alato any other amino acid. 9b). Similar to SMAC/DIABLO. As will be discussed below. This figure was prepared using PyMol (see the online links box) and Molscript124. both in vivo and in vitro66. Hid and Grim proteins. has also been shown to inhibit several caspases.95. HTRA2/OMI. One molecule of p35 binds to each monomer of the caspase-8 dimer. hydrophobic residue in the fourth position (FIG.REVIEWS this bond is protected by the neighbouring N terminus of p35. or on the BIR1 and BIR2 domains of Diap1 (REFS 61. where it interacts with several IAPs and removes the IAP-mediated inhibition of both initiator and effector caspases95. c-IAP1 and c-IAP2. Another fruitfly protein. the founding member of this family is the mitochondrial protein SMAC/DIABLO91. SMAC/DIABLO is released from the intermembrane space of mitochondria into the cytosol. In addition. In contrast to XIAP. However. This observation explains why c-IAP1 and c-IAP2 can bind to. Due to the cleavage of the mitochondria-targeting sequence. a b Reactivesite loop Caspase-8 Cys360 Asp87 Caspase-8 p35 p35 p35 N N-terminal loop Figure 8 | Molecular mechanism of p35-mediated inhibition of caspase-8. there are at least four functional homologues of SMAC/DIABLO: Reaper.94). 80). The active-site conformation of the BIR3-bound caspase-9 closely resembles that of the inactive caspase-9 monomer. The active-site loops are shown in dark blue. of caspase-8. 8b). which has been documented in molecular detail (FIG. which is the active-site residue. the bovine homologue of HTRA2/OMI lacks this motif 101. Cys287 on loop L2. the mature SMAC/DIABLO protein contains a tetrapeptide at its N terminus — Ala-Val-Pro-Ile — which binds to the conserved surface groove on the BIR3 domain of XIAP (REFS 93. The N terminus of p35 restricts solvent access to this intermediate. probably through the disruption of the active site of the enzyme as was shown for the inhibition of serine proteases by serpins89. The RHG proteins bind to and antagonize Diap1-mediated suppression of Dronc and Drice. The binding site for this conserved tetrapeptide motif is the conserved surface groove on the BIR2 and BIR3 domains of XIAP (REFS 93. the N terminus of p35 is translocated into the active site of the caspase85. 9a) and can antagonize XIAP-mediated inhibition of caspase-9 at high concentrations96–100. The thioester intermediate is shown between Asp87 of p35 and Cys360. Covalent inhibition of caspases by viral proteins. During apoptosis. This figure was prepared using Molscript124. but do not inhibit.92. requires an invariant N-terminal Ala residue. the RHG proteins contain an N-terminal IAP-binding tetrapeptide motif (FIG.

In fruitflies. Grim and Sickle in the fruitfly Drosophila melanogaster — that contain a conserved IAP-binding tetrapeptide motif. the procaspase-9 zymogen does not stably interact with .69. The reasons are simple: the binding site for the tetrapeptide motif maps to the surface groove of the BIR2 or BIR3 domain of XIAP (or c-IAP1 or c-IAP2). Removal of effector-caspase inhibition a Vertebrates SMAC/DIABLO Human caspase-9 Mouse caspase-9 Xenopus laevis caspase-9 Human HTRA2/OMI Mouse HTRA2/OMI Fruitflies Reaper Grim Hid Sickle Jafrac2 b AVPIAQKS ATPFQEGL AVPYQEGP ATPVFSGE AVPSPPPA AVPAPPPT Ile Ala Val Phe Pro Ala Thr AVAFYIPD Pro AIAYFLPD AVPFYLPE AIPFFEEE SMAC/DIABLO (AVPI) AKPEDNES bound to BIR3 of XIAP Caspase-9 (ATPF) bound to BIR3 of XIAP Figure 9 | SMAC/DIABLO-mediated removal of caspase-9 inhibition by XIAP.111.107–110. Remarkably. it was anticipated that the underlying mechanisms of caspase regulation should be conserved. Dronc does not contain any sequences that resemble the IAPbinding tetrapeptide motif 69. The IAP-binding tetrapeptide motifs from SMAC/DIABLO (AVPI) and from caspase-9 (ATPF) both bind to the same conserved surface groove on the BIR3 (baculoviral IAP repeat-3) domain of XIAP. Drice is the caspase-3 orthologue in fruitflies. which recruits XIAP to inhibit caspase-9. This figure was prepared using PyMol (see the online links box). Similar to the situation in mammals. In mammals. caspase-9 and HTRA2 (high-temperature-requirement protein A2)/OMI in mammals and the RHG proteins Reaper. which both require physical interactions with IAPs (REF. During apoptosis. respectively. modelling studies indicate that steric clashes preclude XIAP from simultaneously binding to caspase-3 and SMAC/DIABLO77. binding to both the BIR2 and BIR3 domains is required for the SMAC/DIABLO-mediated removal of caspase-7 inhibition111. So. this linker sequence is not 904 | NOVEMBER 2004 | VOLUME 5 ©2004 Nature Publishing Group www. more importantly. Given the extraordinary conservation of the apoptotic pathway between mammals and fruitflies (FIG. Drice regulation by Diap1 and RHG proteins. Subsequent experiments confirmed that this sequence is indeed primarily responsible for the interactions between caspase-9 and XIAP (REF. it is not capable of removing the IAP-mediated inhibition of effector caspases. 10). Importantly. The molecular mechanisms between IAP-mediated regulation of caspase-9 and Dronc are different not only in their mutual recognition but. 9). b | Molecular mechanism of SMAC/DIABLO-mediated removal of caspase-9 inhibition by XIAP (X-linked IAP). 9b). 9b). However. recent biochemical and structural studies have revealed different mechanisms for the regulation of caspases in fruitflies61. This recognition is essential for the Diap1mediated negative regulation of Dronc69. thereby competitively removing caspase-9 (REF.REVIEWS Dronc59. Diap1 functions as an E3 ubiquitin ligase that recognizes and ubiquitylates Dronc69. Further conserved amino acids in fruitflies are shown in blue. Proteolytic cleavage of procaspase-9 after Asp315 results in the exposure of an internal tetrapeptide motif. Although a conclusive mechanism remains elusive. During apoptosis. binding to the BIR domains requires not only the N-terminal tetrapeptide of SMAC/DIABLO. structural analysis revealed that the Dronc-binding surface on the BIR2 domain of Diap1 coincides with that required for binding to the N-terminal sequences of the RHG proteins69 (FIG.nature.102. c-IAP1 or c-IAP2. In mammals. in the way that the caspases are regulated. This model is consistent with the observation that monomeric SMAC/DIABLO mutants only weakly interacted with the BIR2 domain and were unable to remove the IAP-mediated caspase-3 inhibition95. The N-terminal IAP-binding motifs of the RHG proteins bind to a conserved surface groove on the BIR2 domain of Diap1 (REF. Surprisingly. 12). Before proteolytic processing. However. the inhibition of caspase-3 or -7 entails a linker segment that immediately precedes the BIR2 domain of XIAP. Hid. a | The inhibitor of apoptosis (IAP)-mediated inhibition of caspases can be antagonized by a group of proteins — including SMAC (second mitochondria-derived activator of caspases)/DIABLO (direct IAP-binding protein with low pI). biochemical analyses revealed that the BIR2 domain of Diap1 recognizes a 5-amino-acid sequence in the linker region between the prodomain and the caspase unit of Dronc69. Dronc regulation by Diap1 and RHG proteins. XIAP potently inhibits the catalytic activity of caspase-9 (REF. This explains how the RHG proteins competitively eliminate Diap1-mediated negative regulation of Dronc. Intriguingly. where it uses a similar IAP-binding tetrapeptide motif to bind to the BIR3 domain of XIAP (FIG. 81). however. Removal of initiator-caspase inhibition Caspase-9 regulation by XIAP and SMAC/DIABLO.105. 81) (FIG. whereas the RHG proteins can relieve Diap1-mediated suppression of Caspase-3 and -7 regulation by IAPs and SMAC/DIABLO. SMAC/DIABLO in mammals and the RHG proteins in fruitflies antagonize IAP-mediated inhibition of both the initiator and effector caspases through distinct mechanisms. Dronc is the caspase-9 orthologue in fruitflies. Rather. however. Preferred and allowed amino acids are indicated in purple and yellow. 103). thereby targeting Dronc for proteasome-mediated degradation. 70). The tetrapeptide motif has the consensus sequence A-(V/T/I)-(P/A)-(F/Y/I/V/S). The N terminus (Ala-Thr-ProPhe) of the small subunit of caspase-9 conforms to the IAP-binding tetrapeptide motif 81 (FIG. which indicates that Dronc might bind to Diap1 in a way that is similar to the caspase-9–XIAP interactions. In this model. Diap1 directly binds to and suppresses Dronc104–106. Diap1 shows no effect on the catalytic activity of Dronc61. Although the SMAC/DIABLO tetrapeptide in isolation can remove XIAP-mediated caspase-9 inhibition95. whereas the fragment that is responsible for inhibiting caspase-3 or -7 is located between the BIR1 and BIR2 domain of XIAP (or c-IAP1 or c-IAP2). and the fruitfly proteins contain another binding component (conserved residues 5–8). It is this mutual exclusion that allows SMAC/DIABLO to remove the XIAP-mediated inhibition of caspase-9. This steric clash precludes XIAP from simultaneously binding to caspase-7 and SMAC/DIABLO. SMAC/DIABLO is released from mitochondria into the cytoplasm. 2). but also an extensive surface that is available only in the wild-type dimeric SMAC/DIABLO protein77. a conserved IAP-binding motif in caspase-9 and SMAC/DIABLO mediates opposing effects on caspase activity.

and the mechanisms of removal of IAP-mediated inhibition of caspases by a diverse family of proteins that share the IAP-binding tetrapeptide motif. restores the competent conformation of the active site. as exemplified by the limited insights from studies in fruitflies. the mechanisms of IAPand p35-mediated inhibition of caspases.114. the mechanisms that control the regulation of caspases are not. 61). due to the absence of the supporting L2′ loop. Our knowledge of programmed cell death primarily comes from investigations of several model organisms — the nematode. this conserved IAP-binding tetrapeptide motif helps to unify mechanisms of apoptosis regulation from fruitflies to mammals. Cleavage after residue Asp20 of Drice could have another important function. b | A close-up view of the overlapping binding between Dronc and RHG to the same conserved surface groove on the BIR2 domain of Diap1. the case of the initiator caspases seems to be a complicated one. Biochemical analyses showed that Diap1 directly inhibits the catalytic activity of Drice through its BIR1 domain. However. this might be accomplished by the apoptosome by entirely different means28. which results in a dramatic increase (up to 2. both before and after proteolytic processing. fruitflies and mammals. Since Diap1 is readily converted into an N-terminal Drice-inhibiting BIR1 domain and a C-terminal Dronc-targeting domain by the catalytic activity of Dronc61.80. Restoring the competent conformation of the active site is probably important for caspase-9 activation. The fact that caspase-9 is a monomer predicts that its active site would be in an incompetent conformation.115. is present predominantly as a monomer54. respectively54. Caspase-8. the missing L2′ loop is provided. Unfortunately. and that this inhibition can be countered effectively by the RHG proteins61. At present.81). It is important to realize that the conserved IAP-binding tetrapeptide motif has an essential role not only in caspase inhibition. Recent investigations of caspase regulation have led to a significantly improved understanding of the mechanism of activation of effector caspases. The essence of this model is that. Diap1 binds to and inhibits Drice only after the cleavage of its N-terminal 20 amino acids61. we do not yet have a comprehensive understanding of the conserved and divergent mechanisms of caspase regulation across species. Unlike the effector caspases. to the same conserved surface groove on the BIR2 (baculoviral IAP repeat-2) domain of Diap1. These physical differences determine their functional variations. This figure was prepared using PyMol (see the online links box). Interestingly. Grim and Sickle). which could serve as an auto-inhibitory sequence for Diap1 function. However. we do not yet understand the exact molecular mechanism of how Diap1 inhibits Drice. and does not address the mechanism of initiator-caspase activation.115. Activation of initiator caspases. The ‘proximity-induced dimerization’ model121 — which represents a qualitative refinement of the induced-proximity model — argues that the caspase-9 and caspase-8 initiator caspases are activated on dimerization. the accuracy of this model awaits further testing. The apoptotic pathway is conserved among these species. The ‘induced-proximity’ model states that the initiator caspases auto-process themselves when brought into close proximity of each other116. 31. this proposed mechanism might also lead to the removal of the BIR1 domain. Alternatively. Our understanding of how the initiator caspases are activated is far from complete113. Despite these biochemical advances. this model merely describes a process. Crystal structures of BIR1 bound to the RHG peptides show that the RHG proteins use their N-terminal IAP-binding motif to bind to the same surface groove61. but NATURE REVIEWS | MOLECUL AR CELL BIOLOGY ©2004 Nature Publishing Group VOLUME 5 | NOVEMBER 2004 | 9 0 5 . which is facilitated by the apoptosome and DISC oligomeric complexes. At present. which indicates a different mode of inhibition.000-fold) in the catalytic activity of caspase-9 (REF. this. on the other hand. a BIR2 of Diap1 Phe118 Phe5 N N C N b C Hid Dronc Figure 10 | Molecular mechanism of RHG-mediated removal of Dronc suppression by Diap1.113. thereby removing Diap1-mediated inhibition of Drice. for which both the zymogen and the activated enzymes exist as a constitutive homodimer. conserved in Diap1. the proposed N-end rule could facilitate the degradation of Drice as well. Caspase regulation in fruitflies and nematodes. together with the supporting interactions from the dimer interface. on dimerization.REVIEWS also in the removal of caspase inhibition. since Drice forms a stable complex with the BIR1 domain of Diap1 (REF. Hid. which are SMAC/DIABLO (direct inhibitor of apoptosis (IAP)-binding protein with low pI) homologues. however. as the supporting evidence is not definitive. was reported to be present in an equilibrium between monomers and dimers114. The IAPbinding motif of RHG-protein Hid (magenta) and the Dronc peptide (blue) are shown. The frontiers N-END RULE A ubiquitin-dependent pathway that targets proteins for degradation through their destabilizing N-terminal residues. Two models have been proposed. So. to accelerate the degradation of the full-length Diap1 protein through the N-END RULE112. this is accomplished through association with the apoptosome. This model is a succinct summary of experimental observations in which accelerated processing and elevated caspase activity were detected after fusion of the target caspase with a heterologous dimerization domain38.117–120. In cells. a | Mutual exclusion of binding of the fruitfly Drosophila melanogaster caspase-9 homologue Dronc and that of RHG proteins (Reaper. Diap1mediated inhibition of Drice involves a highly conserved surface groove on the BIR1 domain61. that is. Caspase-9. as the purpose of the L2′ loop is to regulate allosterically the active-site conformation. we do not yet understand how caspase-9 is activated by the apoptosome.

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