The protease caspase‑9 (CASP9) is normally present as an inactive monomeric propeptide (procaspase‑9 or zymogen). Upon apoptosis procaspase‑9 (CASP9(1‑416) is recruited to APAF1:cytochrome C (CYCS):ATP complex to form the caspase‑activating apoptosome (Hu Q et al. 2014; Cheng TC et al. 2016). The cryo-EM structures have established that the nucleotide-binding oligomerization domain (NOD) of APAF1 mediates the heptameric oligomerization of APAF1, while its tryptophan-aspartic acid (WD40) domain interacts with CYCS (Yuan S & Akey CW 2013). The caspase recruitment domain (CARD) of APAF1 recruits the N‑terminal CARD of CASP9(1‑416) through homotypic CARD:CARD interactions (Li P et al. 1997; Qin H et al. 1999; Yuan S et al. 2010; Yuan S & Akey CW 2013). These homotypic interaction motifs are thought to interact with each other through three types of interfaces, type I, II, and III, which cooperate to generate homo- and hetero-oligomers from relatively small assemblies to open-ended filaments (Ferrao R & Wu H 2012). Structural and mutagenesis studies showed that all type I, II, and III interfaces are involved in the caspase-9 activation by APAF1-mediated helical oligomerization of CARDs (Hu Q et al. 2014; Cheng TC et al. 2016; Su TW et al. 2017; Li Y et al. 2017). Cryo-EM structure of the holo-apoptosome revealed an oligomeric CARD disk above the heptameric apoptosome ring with estimated molecular ratios between 2-5 zymogens per 7 APAF1 molecules (Hu Q et al. 2014; Cheng TC et al. 2016). The structural and biochemical studies showed that APAF1-CARD and CASP9-CARD initially formed a 1:1 complex in solution, which at higher concentrations is further oligomerized into a 3:3 complex. The 3:3 complex was reported as a core arrangement of the 4:3 or 4:4 APAF1-CARD:CASP9-CARD complex in the helical assembly of the CARD disk (Cheng TC et al. 2016; Su TW et al. 2017; Li Y et al. 2017; Dorstyn L et al. 2018). Thus, APAF1:CASP9 (1-416) heterodimers may be recruted to the assembling apoptosome as part of its activation.
The Reactome event describes the apoptosome assembly with the stoichiometry of 4 procaspase-9 zymogens per 7 APAF1 molecules. The formation of 1:1 and other combinations of APAF1:CASP9(1-416) complexes is not shown.