The atomic-resolution structure of human caspase-8, a key activator of apoptosis

Watt, W.; Koeplinger, Kenneth A.; Mildner, Ana M.; Heinrikson, Robert L.; Tomasselli, Alfredo G.; Watenpaugh, Keith David

doi:10.1016/s0969-2126(99)80180-4

Cited by 167 publications

(158 citation statements)

References 35 publications

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“…The core of the enzyme is formed by a 12-stranded ␤-sheet, which is surrounded by a total of 14 ␣-helices. The overall fold of caspase-2 is similar to that of other caspases for which structures have been determined previously (30,(32)(33)(34)(35)(36)(37)(38)(39). This is illustrated by a superposition of the C␣ chains with caspase-3, another representative of the specificity group II caspases, and caspase-9, the closest relative of caspase-2 based on sequence identity (Fig.…”

Section: Resultssupporting

confidence: 71%

Crystal Structure of Caspase-2, Apical Initiator of the Intrinsic Apoptotic Pathway

Schweizer

Briand

Grütter

2003

Journal of Biological Chemistry

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The cell death protease caspase-2 has recently been recognized as the most apical caspase in the apoptotic cascade ignited during cell stress signaling. Cytotoxic stress, such as that caused by cancer therapies, leads to activation of caspase-2, which acts as a direct effector of the mitochondrion-dependent apoptotic pathway resulting in programmed cell death. Here we report the x-ray structure of caspase-2 in complex with the inhibitor acetyl-Leu-Asp-Glu-Ser-Asp-aldehyde at 1.65-Å resolution. Compared with other caspases, significant structural differences prevail in the active site region and the dimer interface. The structure reveals the hydrophobic properties of the S5 specificity pocket, which is unique to caspase-2, and provides the details of the inhibitorprotein interactions in subsites S1-S4. These features form the basis of caspase-2 specificity and allow the design of caspase-2-directed ligands for medical and analytical use. Another unique feature of caspase-2 is a disulfide bridge at the dimer interface, which covalently links the two monomers. Consistent with this finding, caspase-2 exists as a (p19/p12) 2 dimer in solution, even in the absence of substrates or inhibitors. The intersubunit disulfide bridge stabilizes the dimeric form of caspase-2, whereas all other long prodomain caspases exist as monomers in solution, and dimer formation is driven by ligand binding. Therefore, the central disulfide bridge appears to represent a novel way of dimer stabilization in caspases.

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Section: Resultssupporting

confidence: 71%

Crystal Structure of Caspase-2, Apical Initiator of the Intrinsic Apoptotic Pathway

Schweizer

Briand

Grütter

2003

Journal of Biological Chemistry

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“…The purification protocol was adapted from published procedures 25 and was described previously. 22 Cells were harvested, resuspended in PBS buffer and lyzed by french press.…”

Section: Methodsmentioning

confidence: 99%

“…The recently determined solution structure of procaspase-8 and the available crystal structures of caspase-8 in the presence of various inhibitors provide important information to decipher the activation mechanism of caspase-8. [22][23][24][25] Additional mutational studies dissected the interplay between dimerization and proteolytic cleavage. [13][14][15]22,[26][27][28] Although dimerization is required and by itself sufficient for caspase-8 activity, cleavage of the inter-subunit linker at two different recognition motifs is important for dimer stabilization and is, thus, essential for caspase-8 activity.…”

mentioning

confidence: 99%

Studies of the molecular mechanism of caspase-8 activation by solution NMR

2009

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Caspases are the key players of apoptosis and inflammation. They are present in the cells as latent precursors, procaspases, and are activated upon an apoptotic or inflammatory stimulus. The activation mechanism of caspases has been studied extensively by biochemical and biophysical methods. Additional structural information on active caspases with a variety of different inhibitors bound at the active site is available. In this study, we investigated the cleavage mechanism of caspase-8 from its zymogen to active caspase-8 by solution NMR and by biochemical methods. The intermolecular cleavage reaction using the catalytically inactive C285A procaspase-8 mutant is triggered by adding caspase-8 and followed by 15 N, 1 H-NMR spectroscopy. The spectrum that initially resembles the one of procaspase-8 gradually over time changes to that of caspase-8, and disappearing peaks display exponential decaying intensities. Removal of either one of the cleavage recognition motifs in the linker, or phosphorylation at Tyr380, is shown to reduce the rate of the cleavage reaction. The data suggest that dimerization repositions the linker to become suitable for intermolecular processing by the associated protomer. Furthermore, analysis of inhibitor binding to the active caspase-8 reveals an induced-fit mechanism for substrate binding.

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“…Although the protease domains of caspases-3 and -8 are structurally quite similar, 22,23,33 several regions of the proteins are not conserved. Procaspase-8 contains N-terminal tandem DEDs, which provide interaction surfaces with adapter proteins and death receptors, 12,34 while procaspase-3 contains a short pro-domain (28 amino acids) in an extended structure.…”

Section: Discussionmentioning

confidence: 99%

“…1(A)], the dimer interfaces are significantly different, particularly in the central strand, b-strand 8. 22,23 The central b-strand of caspase-8 has limited, longer range, interactions between backbone atoms of F468 and P466' (3.6 Å ) and P466 and T467' (3.3 Å , backbone to side-chain or 3.9 Å , backbone to backbone) [see Fig. 1(B)].…”

Section: Introductionmentioning

confidence: 99%

Redesigning the procaspase‐8 dimer interface for improved dimerization

MacKenzie

Clark

2014

Protein Science

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Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.

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The atomic-resolution structure of human caspase-8, a key activator of apoptosis

Cited by 167 publications

References 35 publications

Crystal Structure of Caspase-2, Apical Initiator of the Intrinsic Apoptotic Pathway

Crystal Structure of Caspase-2, Apical Initiator of the Intrinsic Apoptotic Pathway

Studies of the molecular mechanism of caspase-8 activation by solution NMR

Redesigning the procaspase‐8 dimer interface for improved dimerization

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