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

Abstract

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.

Structures of BIRC6-client complexes provide a mechanism of SMAC-mediated release of caspases

“…4, C to E). This hypothesis has recently been substantiated by the fact that charge-reversal mutations in the CBM loop impair BIRC6-mediated inhibition of casp-3 and ubiquitylation of casp-7 ( 31 ).…”

“…1A). BIRC6 interacts with and inhibits casp-3, casp-7, and casp-9, and these interactions are inhibited by SMAC ( 23 , 31 ). BIRC6 has also been established as a substrate for caspases as well as HTRA2 (Fig.…”

BIRC6 Modulates the Protein Stability of Axin to Regulate the Growth, Stemness, and Resistance of Renal Cancer Cells via the β-Catenin Pathway

“…There are eight identified BIRC proteins, most of which provide protection against or delay in cell death induced by apoptotic stimuli when expressed at high levels . Additionally, most of these proteins can bind to specific caspases to mediate the antiapoptosis effects . BIRC6 is a member with the largest molecular weight and is the only one known to be linked to the membrane so far .…”

“…Baculoviral IAP repeat-containing 6 (BIRC6) is a member of the inhibitor of apoptosis proteins family and contains a ubiquitin-conjugating enzyme E2 structure domain . BIRC6 possesses both ubiquitin-conjugating enzyme activity and apoptosis-inhibiting function, impeding cell apoptosis by facilitating the ubiquitination and degradation of targeted apoptotic proteins . As an apoptosis inhibitor, BIRC6 plays a significant role in the development of malignancies .…”

A conserved N-terminal motif of CUL3 mediates assembly and licenses E3 ligase activation of CRL3KLHL22

“…For example, the dimerization of CRL1 Fbx4 is required for substrate binding and E3 ligase activation 39 . Furthermore, the giant E3 ubiquitin ligase BIRC6 [baculoviral IAP repeat (BIR) domain-containing proteins 6] is assembled and functions as dimer [40][41][42][43] . The dimeric architecture of BIRC6 is essential for binding of its substrates, including SMAC (Second mitochondria-derived activator of caspases) and the serine protease HTRA2 (High temperature requirement protein A2).…”