Structural basis for SMAC-mediated antagonism of caspase inhibition by the giant ubiquitin ligase BIRC6

Certain Inhibitors of apoptosis (IAP) family members are sentinel proteins preventing untimely cell death by inhibiting caspases. Antagonists including second mitochondria-derived activator of caspase (SMAC) regulate IAPs, driving cell death. Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP with dual E2/E3 ubiquitin ligase activity, regulates programmed cell death through unknown mechanisms. We show BIRC6 directly restricts executioner caspases-3 and -7, and ubiquitinates caspases-3, -7 and -9 working exclusively with non-canonical E1, UBA6. Importantly, we show SMAC suppresses both mechanisms. Cryo-electron microscopy structures of BIRC6 alone and in complex with SMAC reveals BIRC6 is an anti-parallel dimer juxtaposing the substrate-binding module against the catalytic domain. Furthermore, we discover SMAC multi-site binding to BIRC6 results in a sub-nanomolar affinity interaction, enabling SMAC to competitively displace caspases thus antagonizing BIRC6 anti-caspase function.