Structural Investigation Of Drosophila Dark Apoptosome

The execution of apoptosis is performed by cascade activated caspases, the activated caspases digest substrates in the living cells. The initiator caspase, as caspase-9 in mammals or its counterpart Dronc in fruit flies, is activated in an auto-catalytical manner, which is facilitated by a multimeric adaptor protein complex known as apoptosome. The atomic resolution structure of apoptosome from higher organisms remains unknown, and so as the mechanism of how initiator caspase as caspase-9 or Dronc is activated by the apoptosomes.Dark protein from model organism Drosophila melanogatster, is the orthologue of Apaf-1 from mammals. The previous studies shows that, Dark protein exists as a monomer state in solution, in the presence of dATP, Dark oligomerized to form a big complex, namely, the apoptosome, to facilitate the activation of initiator caspase Dronc.The purpose of our study is to elucidate the mechanism of how Dark apoptosome facilitate the activation of caspase Dronc in the Drosophila apoptosis pathway.Finally, we determined the cryo-electron microscopic structure of the Dark apoptosome at a near-atomic resolution of 4.0 ?. The structure shows that the 2.5 MDa Dark apoptosome composed by 16 molecules of Dark monomers, contains two octamer rings. The assembly pattern between the Dark protomers share some similarity with CED-4 apoptosome from C.elegans.In the meanwhile, we found that Dronc itself could potently induces the oligomerization of Dark proteins in the absence of dATP, within the Dronc is efficiently activated. Initiator caspases interact with apoptosome protein through ammino-terminal CARD domains. We also determined the cryo-EM structure of the Dark apoptosome bound to Dronc-CARD at a resolution of 4.1 ?. The structure shows that 16Dronc-CARD molecules also composed into two rings that stacked between two octameric rings of the Dark proteins. The Dronc-CARD interacts with nearby Dark protein specifically at CARD domain and the WD40 repeats. These interactions are indispensable for the activation of the Drosophila initiator caspase Dronc.