Structures of BIRC6-Client Complexes Provide Mechanism of Smac-Mediated Release of Caspases

Apoptosis is tightly regulated and essential for metazoan development1, 2. Excessive apoptosis contributes to neurodegenerative disease, while diminished apoptosis can lead to inflammation and cancer3. Inhibitor of apoptosis (IAP) proteins are the principal actors that restrain apoptotic activity and are thus attractive therapeutic targets4. IAPs in turn are regulated by mitochondria-derived pro-apoptotic factors such as Smac and HtrA24. Here, through a series of cryo-electron microscopy (cryo-EM) structures of full-length baculoviral IAP repeat-containing protein 6 (BIRC6) bound to Smac, caspase-3, caspase-7 and HtrA2, we provide the molecular basis for BIRC6-mediated caspase inhibition and its release by Smac. We demonstrate that BIRC6 cooperates preferentially with the non-canonical E1 enzyme UBA6 to ubiquitylate caspases and that caspase ubiquitylation is effectively inhibited by Smac through near-irreversible interactions. The dimeric arrangement of BIRC6 resolves the long-standing question of how the evolutionarily conserved single-BIR domain IAPs sequester caspases and how Smac binding antagonizes IAPs. This collection of BIRC6 structures provides critical insights into IAP-mediated apoptosis regulation, relevant for the development of current and future apoptosis-targeting cancer therapeutics.