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Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9

2020; Elsevier BV; Volume: 180; Linguagem: Inglês

10.1016/j.biochi.2020.10.013

1638-6183

Ali-Reza Noori, Amin Tashakor, Maryam Nikkhah, Leif A. Eriksson, Saman Hosseinkhani, Howard O. Fearnhead,

ATP Synthase and ATPases Research

Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1–921) on Apaf-1 interactions and caspase activation. Apaf-1 1–921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.