| Summary: | Apoptosis, a form of genetically programmed cell death, can be triggered by either internal or external signals ultimately activating caspases, a family of proteases1. Certain members of the inhibitors of apoptosis (IAP) family are sentinel proteins preventing untimely cell death by inhibiting caspases. IAPs are in turn regulated by antagonists including second mitochondria-derived activator of caspase (SMAC). Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP, possesses dual E2/E3 ubiquitin ligase activity and is implicated in apoptosis via caspase inhibition2–7. How this is achieved remains unknown. Here we show BIRC6 directly restricts activated caspase-3, and ubiquitinates activated caspases-3, −7 and −9 working exclusively with the non-canonical E1, UBA6. Importantly, we show SMAC supresses both mechanisms. Cryo-electron microscopy (cryo-EM) structures of BIRC6 alone and in complex with SMAC reveal BIRC6 exists as an anti-parallel dimer with a substrate-binding module juxtaposed to the catalytic domain at each end, and we identify multiple highly conserved unannotated domains important for architecture and function. Through our structural, biochemical and biophysical findings, we discover SMAC engages BIRC6 at multiple sites resulting in a sub-nanomolar affinity enabling SMAC to competitively displace caspases, thus antagonising BIRC6-mediated caspase inhibition.
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