page_title: Apoptosome
section_title: Structure
hierarchical_section_title: Apoptosome / Structure
caption_attribution_description: English: Space-fill drawing comparing the structures of the apoptosome molecular assemblies from human (Apaf-1), fruit fly (Dark), and nematode (CED4). The apoptosome subunits (shown in blue and purple) assemble when needed, to communicate between the system that signals cell death, such as cytochrome C for humans (in orange), to the system that disassembles the cell, such as the caspase proteases for humans, which are activated by binding to the CARD domains of the apoptosome (in purple). Image drawn by David Goodsell, from PDB files 3J2T, 3IZ8, and 3LQQ.
context_page_description: The apoptosome is a large quaternary protein structure formed in the process of apoptosis. Its formation is triggered by the release of cytochrome c from the mitochondria in response to an internal or external cell death stimulus. Stimuli can vary from DNA damage and viral infection to developmental cues such as those leading to the degradation of a tadpole's tail.
  In mammalian cells, once cytochrome c is released, it binds to the cytosolic protein Apaf-1 to facilitate the formation of an apoptosome. An early biochemical study suggests a two-to-one ratio of cytochrome c to apaf-1 for apoptosome formation. However, recent structural studies suggest the cytochrome c to apaf-1 ratio is one-to-one. It has also been shown that the nucleotide dATP as third component binds to apaf-1, however its exact role is still debated. The mammalian apoptosome had never been crystallized, but a human APAF-1/cytochrome-c apoptosome has been imaged at lower resolution by cryogenic transmission electron microscopy in 2002, revealing a heptameric wheel-like particle with 7-fold symmetry.
context_section_description: The apoptosome is a multimolecular holoenzyme complex assembled around the adaptor protein Apaf1 (apoptotic protease activating factor 1) upon mitochondria-mediated apoptosis which must be stimulated by some type of stress signal T formation of the apoptosome requires the presence of ATP/dATP and cytochrome c in the cytosol.
  A stress stimulus can trigger the release of cytochrome c into the cytoplasm which will then bind to the C-terminus of Apaf-1 within a region containing multiple WD-40 repeats. The oligomerization of Apaf-1 appears to be accompanied by synchronized recruitment of procaspase-9 to the CARD motif at the Apaf-1 N-terminus. The apoptosome triggers the activation of caspases in the intrinsic pathway of apoptosis.
  The wheel-shaped heptameric complex with sevenfold symmetry structure of the apoptosome was first revealed at 27 Å resolution by electron cryomicroscopy techniques and has a calculated mass of about 1 MDa (Acehan et al. 2002). This wheel-like particle has seven spokes and a central hub. The distal region of the spoke has a pronounced Y shape. The hub domain is connected to the Y domain by a bent arm. Each Y domain comprises two lobes (a large one and a small one) between which cytochrome c binding sites. Because the resolution of the apoptosome structure was relatively low, two controversial models for apoptosome assembly were proposed. One model suggests NOD domains form the central hub and the CARD domains form a freer ring at the top of the NOD region. Another model proposes that Apaf-1 is organized in an extended fashion such that both the N-terminal CARD and the nucleotide binding region form the central hub of the apoptosome, whereas the 13 WD-40 repeats constitute the two lobes. The large lobe is formed by seven repeats and the small lobe is formed by six repeats. Each caspase- 9 molecule binds a CARD domain at the central hub, forming a dome shaped structure. This controversy has been resolved by a recent high resolution structure of the human apoptosome-procaspase-9 CARD complex. This structure clearly demonstrated that only the NOD regions form the central hub of the apoptosome (see pictures), while CARD is flexibly linked to the platform of apoptosome and becomes disordered in the ground state apoptosome. Once apoptosome binds to procaspase-9, the Apaf-1 CARDs and procaspase-9 CARDs form a flexible disk-like structure sitting above the platform. The number of WD-40 repeats has also been proved to be 15 instead of 13, and it is composed of a 7-bladed beta-propeller and an 8-bladed beta-propeller.
  Evidence from Wang and colleagues indicates that the stoichiometric ratio of procaspase-9 to Apaf-1 within the complex is approximately 1:1 . This was further proved by quantitative mass spectrometry analysis. The stoichiometry of cytochrome c to Apaf-1 within the complex is proved to be 1:1. There are some debates about whether stable incorporation of cytochrome c into the apoptosome is required following oligomerization, but recent structural data favor the idea that cytochrome c stabilizes the oligomeric human apoptosome. However, cytochrome c may be not required for the assembly of apoptosome in non-mammalian species, such as worms and fruit flies. In addition, several other molecules, most notably caspase-3, have been reported to co-purify with the apoptosome and caspase-3 has been proved to be able to bind the apoptosome-procaspase-9 complex.
  Apaf-1 forms the backbone of the apoptosome. It has three distinct regions: the N-terminal caspase-recruitment domain (CARD, residues 1–90), a central nucleotide-binding and oligomerization region (NB-ARC/NOD, 128–586) and a C-terminal WD40 region (613–1248) making up a protein about 140 KDa.
  The CARD domain of Apaf-1 interacts with procaspase-9 and involved with recruitment within the apoptosome.
  The NB-ARC/NOD region exhibits significant sequence similarity to the C. elegans Ced-4 protein.
  The C-terminal WD40 region of Apaf1 contains 15 WD-40 repeats structured into two b-propeller-shaped domains. WD-40 repeats are sequences around 40 amino acids lo
caption: Human, fruit fly, & nematode apoptosomes
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