Bcl2 Family Of Proteins

The Bcl-2 family of proteins controls the mitochondria-initiated intrinsic apoptosis and regulates the death receptor-initiated extrinsic cell death. More than two dozen Bcl-2 family proteins have been identified in multicellular organisms examined to date (12) (see Chapter 2). On the basis of function and sequence similarity, the diverse Bcl-2 members can be grouped into three subfamilies. The anti-apoptosis subfamily, represented by Bcl-2/Bcl-XL in mammals and CED-9 in worms, inhibits programmed cell death by distinct mechanisms. For example, Bcl-2/Bcl-XL functions by preventing the release of mitochondrial proteins, whereas CED-9 binds CED-4 and prevents CED-4-mediated activation of CED-3. The pro-apoptosis proteins constitute two subfamilies, represented by Bax/Bak and Bid/Bim. Members in the Bcl-2/Bcl-XL subfamily contain all four conserved Bcl-2 homology domains (BH4, BH3, BH1, and BH2), whereas the Bax/Bak subfamily lack the BH4 domain and the Bid/Bim subfamily only contain the BH3 domain. Most members of the Bcl-2 family contain a single membrane-spanning region at their C-termini. Members of the opposing subfamilies as well as between the two pro-apoptotic subfamilies can dimerize, mediated by the amphipathic BH3 helix.

The first structure of the Bcl-2 family of proteins was determined on Bcl-XL by both X-ray crystallography and NMR spectroscopy (13). This structure reveals two centrally located hydrophobic a helices (a5 and a6), packed by five amphipathic helices on both sides (Fig. 2A). Interestingly, the Bcl-XL structure closely resembles the pore-forming domains of bacterial toxins such as diphtheria toxin. This similarity raised an interesting hypothesis that the Bcl-2 family of proteins may form pores at the outer mitochondrial membrane to regulate ion exchange. Indeed, this conjecture has been proven for nearly all members of the Bcl-2 family examined in vitro. However, it is unclear whether the pH-dependent ion-conducting property of Bcl-2 proteins occurs in vivo and, if so, how it contributes to the regulation of apoptosis.

Because most members of the Bcl-2 family associate with lipid membranes using their C-terminal transmembrane region, it is important to examine the structure of membrane-associated Bcl-2 family proteins. Unfortunately, no such structure is yet available due to technical difficulty. Towards this ultimate goal, Bcl-XL was characterized in detergent micelles and, compared to the aqueous solution, exhibited significant structural difference (14).

Bcl-XL or Bcl-2 interacts with the BH3-only subfamily of Bcl-2 proteins such as Bad, Bim, and Bid. The recognition mode was revealed by the solution structure of Bcl-XL bound to a BH3 peptide from Bak (15) (Fig. 2B). The BH3 peptide forms an amphipathic a helix and interacts with a deep hydrophobic groove on the surface of Bcl-XL. The binding of the BH3 domain causes a significant conformational change in Bcl-XL, including the melting of a short a-helix (a3).

The structures of the other two groups of Bcl-2 family members have also been determined. Bid, containing only the BH3 domain and displaying very weak sequence homology with Bcl-XL, exhibits a conserved structure with that of Bcl-XL (16,17). The minor differences include the length and relative orientation of several helices as well as an extra a helix between helices a1 and a2 (Fig. 2C). On the basis of the structure, a model was proposed to explain how the caspase-8-mediated cleavage of Bid (after residue 59) improves its pro-apoptotic function. In this mechanism, removal of the N-terminal 59 amino acids leads to the exposure of the BH3 domain, which mediates binding to the other two subfamilies of Bcl-2 proteins.

Similar to Bid, the structure of the full-length Bax in aqueous solution closely resembles that of Bcl-XL (18). Intriguingly, the C-terminal membrane-spanning region folds back to bind a hydrophobic groove that normally accommodates the BH3 domain of another Bcl-2 protein (Fig. 2D). Thus this structure may represent the inactive or closed form of the Bax/Bak group and suggests a regulatory role for their C-termini in the absence of apoptotic stimuli.

Bax and Bak exist as monomers in aqueous solution but can form homo-oligomers in the presence of detergents. These large homo-oligomers are thought to form channels with a pore size large enough to allow passage of proteins such as cytochrome c, though direct biochemical and structural evidence

Fig. 2. Structure of the Bcl-2 family proteins. (A) Structure of Bcl-XL. The flexible loop linking helices a1 and a2 are represented by a dotted line. (B) Structure of Bcl-XL bound to a BH3 peptide from Bad. This Bad peptide exists as an amphipathic helix, with the hydrophobic side binding to Bcl-XL. (C) Structure of the uncleaved form of Bid. Cleavage after Asp59 results in the activation of Bid, presumably due to exposure of the BH3 helix. (D) Structure of the full-length Bax. Note that the C-terminal amphipathic helix folds back to bind a hydrophobic surface groove, resembling the Bcl-xL-bound Bad peptide.

Fig. 2. Structure of the Bcl-2 family proteins. (A) Structure of Bcl-XL. The flexible loop linking helices a1 and a2 are represented by a dotted line. (B) Structure of Bcl-XL bound to a BH3 peptide from Bad. This Bad peptide exists as an amphipathic helix, with the hydrophobic side binding to Bcl-XL. (C) Structure of the uncleaved form of Bid. Cleavage after Asp59 results in the activation of Bid, presumably due to exposure of the BH3 helix. (D) Structure of the full-length Bax. Note that the C-terminal amphipathic helix folds back to bind a hydrophobic surface groove, resembling the Bcl-xL-bound Bad peptide.

is lacking. Despite structural information on all three subfamilies of Bcl-2 members, how these proteins regulate apoptosis remains largely unknown. Biophysical and structural characterization of the Bcl-2 protein complexes under membrane-like conditions will likely reveal some surprising insights, although it is also possible that other important regulators of Bcl-2 proteins are yet to be identified.

CASPASES: EXECUTIONERS OF APOPTOSIS

Caspases are a family of highly conserved cysteine proteases that cleave after an aspartate in their substrates (19). The critical involvement of a caspase in apoptosis was first documented in 1993 (20), in which CED3 was found to be indispensable for the programmed cell death in the nematode Caenorhabditis elegans. Since then, compelling evidence has demonstrated that the mechanism of apoptosis is evolutionarily conserved, executed by caspases from worms to mammals. At least 14 distinct mammalian caspases have been identified (21) (see Chapter 1).

Caspases involved in apoptosis are divided into two groups: the initiator caspases, which include caspases-1, -2, -8, -9, and -10; and the effector caspases, which include caspases-3, -6, and -7. An

Fig. 3. Structural features of caspases. (A) A representative structure of the inhibitor-bound caspase-3 (PDB code 1DD1). The bound peptide inhibitor is shown in black. The four surface loops that constitute the catalytic groove of one hetero-dimer are labeled. The apostrophe denotes the other hetero-dimer. Note that L2' stabilizes the active site of the adjacent hetero-dimer. The substrate-binding groove is schematically shown above. (B) The active-site conformation of all known caspases is conserved. Of the four loops, L1 and L3 are relatively constant while L2 and L4 exhibit greater variability. The catalytic residue Cys is shown in black. Two perpendicular views are shown.

Fig. 3. Structural features of caspases. (A) A representative structure of the inhibitor-bound caspase-3 (PDB code 1DD1). The bound peptide inhibitor is shown in black. The four surface loops that constitute the catalytic groove of one hetero-dimer are labeled. The apostrophe denotes the other hetero-dimer. Note that L2' stabilizes the active site of the adjacent hetero-dimer. The substrate-binding groove is schematically shown above. (B) The active-site conformation of all known caspases is conserved. Of the four loops, L1 and L3 are relatively constant while L2 and L4 exhibit greater variability. The catalytic residue Cys is shown in black. Two perpendicular views are shown.

initiator caspase invariably contains an extended N-terminal prodomain (>90 amino acids) important for its function, whereas an effector caspase contains only 20-30 residues in its prodomain sequence. All caspases are synthesized in cells as catalytically inactive zymogens and must undergo proteolytic activation. The activation of an effector caspase, such as caspases-3 or -7, is performed by an initiator caspase, such as caspase-9, through internal cleavages to separate the large and small subunits. The initiator caspases, however, are auto-activated under apoptotic conditions.

The first caspase structure was determined on caspase-1 (or ICE, interleukin-ip-converting enzyme) bound with a covalent peptide inhibitor (22,23). Structural information is now available on caspase-3 (24,25), caspase-7 (26), caspase-8 (27,28), and more recently, caspase-9 (29). In each case, caspase is bound to a synthetic peptide inhibitor (Fig. 3A). These structures reveal that the functional caspase unit is a homo-dimer, with each monomer comprising a large (~20 kDa) and a small (~10 kDa) subunit. Homo-dimerization is mediated by hydrophobic interactions, with six anti-parallel P-strands from each monomer forming a single contiguous 12-stranded P-sheet (Fig. 3A). Five a helices and five short 6 strands are located on either side of the central 6-sheet, giving rise to a globular fold. The active sites, highly conserved among all caspases and located at two opposite ends of the P-sheet, are formed by four protruding loops (Li, L2, L3, and L4) from the scaffold.

Caspases recognize at least four contiguous amino acids, named P4-P3-P2-P1, in their substrates, and cleave after the C-terminal residue (Pi), usually an Asp. The binding sites for P4-P3-P2-P1 are named S4-S3-S2-S1, respectively, in caspases. These sites are located in the catalytic groove. The Li and L4 loops constitute two sides of the groove (Fig. 3). Loop L3 and the following P-hairpin, collectively referred to as L3, is located at the base of the groove. Loop L2, which harbors the catalytic residue Cys, is positioned at one end of the groove with Cys poised for binding and catalysis. These four loops, of which L1 and L3 exhibit conserved length as well as composition, determine the sequence specificity of the substrates.

The S1 and S3 sites are nearly identical among all caspases. The P1 residue (Asp) is coordinated by three invariant residues at the S1 site, an Arg from the L1 loop, a Gln at the beginning of the L2 loop, and an Arg at the end of the L3 loop. The Arg residue on the L3 loop also coordinates the P3 residue (Glu). The S2 and S4 sites are coordinated mainly by the L3 and L4 loops. Because the sequence of L4 is most divergent among caspases, the P2 and P4 residues exhibit greater sequence variation. For example, the L4 loop in caspases-1, -8, or -9 is considerably shorter than that in caspase-3 or -7, resulting in a shallower substrate-binding groove. This observation is consistent with a bulky hydrophobic residue as the preferred P4 residue for caspases-1, -8, or -9.

The conformational similarity at the active site is extended to surrounding structural elements. In particular, loops L4 and L2 from one catalytic subunit are stabilized by the N-terminus (loop L2') of the small subunit of the other catalytic subunit, forming the so-called "loop-bundle" (30).

Most structural information is derived from the inhibitor-bound caspases, which share the same topology at the active site. These observations give the impression that the substrate-binding grooves of caspases are pre-formed. However, in the structure of the free caspase-7 (30), these loops are flexible and quite different from those in the inhibitor-bound caspase-7 (Fig. 4), suggesting that the inhibitor-bound state is transient and trapped by the covalent peptide inhibitors. Thus, substrate binding and catalysis may be a process of induced-fit, accompanied by some large conformation changes, such as the back-and-forth flipping of the critical L2' loop.

Why are procaspase zymogens (except procaspase-9) catalytically inactive? The answer was partially provided by the crystal structure of procaspase-7 (30,31), which reveals significant conformational changes in the four active site loops (Fig. 4). Except L1, all three other loops move away from their productive positions, unraveling the substrate-binding groove. Most notably, the loop-bundle seen in the inhibitor-bound caspases is missing in the procaspase-7 zymogen as the L2' loop is flipped by 180 degrees, existing in a "closed" conformation. This closed conformation is locked by the unprocessed nature of the procaspase-7 zymogen.

The ability of L2' to move freely in response to inhibitor/substrate binding is a decisive feature for the active caspase-7. This ability is acquired through activation cleavage after Asp198 in procaspase-7. Because L2' is at the N-terminus of the small subunit, inverting the order of primary sequences of the large and small subunits could free L2' and hence constitutively activate caspases. Indeed, this prediction was confirmed for caspases-3 and -6 (32) as well as for the Drosophila caspase drICE (33).

In contrast to most other caspases, procaspase-9 exhibits a basal level of activity prior to proteolytic activation (34). The surprising feature may be explained by the fact that procaspase-9 contains an expanded L2 loop, which could allow enough conformational flexibility such that procaspase-9 does not need an inter-domain cleavage to have the L2' loop move to its productive conformation.

inhibitors of apoptosis (iap)

The inhibitor of apoptosis (IAP) family of proteins, originally identified in the genome of baculovirus, suppress apoptosis by interacting with and inhibiting the enzymatic activity of mature caspases (35) (see Chapter 3). At least eight distinct mammalian IAPs including XIAP, c-IAP1, c-IAP2, and ML-IAP/Livin, have been identified, and they all have anti-apoptotic activity in cell culture. A structural feature common to all IAPs is the presence of at least one BIR (baculoviral IAP repeat) domain, characterized by a conserved zinc-coordinating Cys/His motif (CX2CX16HX6C). Some IAPs, such as XIAP and ML-IAP/Livin, also contain a C-terminal RING finger, a C3HC4-type

Fig. 4. Mechanisms of procaspase-7 activation and substrate binding. (A) Structure of an active and free caspase-7 (PDB code 1K88). The active-site loops are flexible. Despite an inter-domain cleavage, the L2' loop still exists in the closed conformation, indicating an induced-fit mechanism for binding to inhibitors/ substrates. (B) Structure of a procaspase-7 zymogen (PDB code 1K86). Compared to that of the inhibitor-bound caspase-7, the conformation of the active site loops does not support substrate-binding or catalysis. The L2' loop, locked in a closed conformation by covalent linkage, is occluded from adopting its productive and open conformation. (C) Comparison of the conformation of the active site loops. Compared to the procaspase-7 zymogen or the free caspase-7, the L2' loop is flipped 180o in the inhibitor-bound caspase-7 to stabilize loops L2 and L4.

Fig. 4. Mechanisms of procaspase-7 activation and substrate binding. (A) Structure of an active and free caspase-7 (PDB code 1K88). The active-site loops are flexible. Despite an inter-domain cleavage, the L2' loop still exists in the closed conformation, indicating an induced-fit mechanism for binding to inhibitors/ substrates. (B) Structure of a procaspase-7 zymogen (PDB code 1K86). Compared to that of the inhibitor-bound caspase-7, the conformation of the active site loops does not support substrate-binding or catalysis. The L2' loop, locked in a closed conformation by covalent linkage, is occluded from adopting its productive and open conformation. (C) Comparison of the conformation of the active site loops. Compared to the procaspase-7 zymogen or the free caspase-7, the L2' loop is flipped 180o in the inhibitor-bound caspase-7 to stabilize loops L2 and L4.

Fig. 5. Structure of the BIR domains. (A) Structure of the BIR2 domain of XIAP. The bound zinc atom as well as the four conserved Cys/His residues are labeled. (B) Superposition of the structure of XIAP-BIR2 with that of human survivin.

zinc-binding module. Most mammalian IAPs have more than one BIR domain, with the different BIR domains exhibiting distinct functions. For example, in XIAP, c-IAP1, and c-IAP2, the third BIR domain (BIR3) potently inhibits the activity of processed caspase-9, whereas the linker region between BIR1 and BIR2 selectively targets active caspase-3. The RING fingers were found to exhibit ubiquitin ligase (E3) activity, which may regulate self-destruction or degradation of active caspases through the 26S proteasome pathway.

The structures of various BIR domains, determined by both NMR and X-ray crystallography (3638), reveal a highly conserved topology, with a three-stranded anti-parallel P sheet and four a helices (Fig. 5A). The three cysteine and one histidine residues, invariant in all BIRs, coordinate a zinc atom. Although the BIR2 and BIR3 domains of XIAP share an identical fold, structure-based mutational analysis revealed that different regions are involved in the interaction with and the inhibition of caspases-3 and -9 (36,37). Several amino acids in the linker sequence preceding BIR2 were found to be essential in targeting caspase-3, while residues on the surface of XIAP-BIR3 inhibited caspase-9.

The smallest IAP is survivin, with only one BIR domain and a C-terminal acidic stretch. In contrast to the relatively stable expression levels of other IAPs, expression of survivin oscillates with cell cycle and peaks at the G2/M phase (39). Recombinant survivin does not inhibit caspase activity in vitro and appears to play an important role in mitosis. Although the structures of survivin reveal that the BIR domain adopts the canonical fold (Fig. 5B), two contrasting modes of dimerization were proposed (40-42), each with supporting evidence.

caspase-iap complex

The IAP-bound structures were determined for two highly conserved effector caspases, caspases-3 and -7 (43-45) (Fig. 6A,B). In the structures, the linker peptide N-terminal to XIAP-BIR2 forms highly similar interactions with both caspases-3 and -7 (Fig. 6C). Compared to the covalent peptide inhibitors, the linker segment of XIAP occupies the active site of caspases, resulting in a blockade of substrate entry. Four residues from the XIAP linker peptide, Gly144-Val146-Val147-Asp148, occupy the corresponding positions for the P1-P2-P3-P4 residues of the substrates, respectively (Fig. 6D). The P1 position is occupied by the N-terminal Gly144 of these four residues. Thus this orientation is the reverse of that observed for the tetrapeptide caspase inhibitors, in which the P1 position is occupied by the C-terminal Asp. Interestingly, despite a reversal of relative orientation, a

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