Apoptosome - Structure

Structure

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 cryomicroscopytechniques 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 a 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 long which end in Trp-Asp and are typically involved in protein–protein interaction.

A short linker and nucleotide binding a/b domains (NBD) that contain conserved Walker boxes A (p-loop 155-161) and B (239-243) follow the N-terminal CARD domain. The Walker boxes A/B are critical for dATP/ATP and Mg2+ binding. Following the NBD is a small helical domain (HD1), a second linker and a conserved winged helix domain (WHD). The NOD region comprises NBD, HD1 and WHD, creating an ATPase domain that is part of the AAA+ family of ATPases. There is a super helical domain (HD2) present in the junction between the NOD and the WD-40 repeats. The WD40 repeats are in groups of eight and seven with linkers connecting them.