The functional domains for Bax∆2 aggregate-mediated caspase 8-dependent cell death

•Disruption of helix α1 makes Baxα mimic Bax∆2 behavior.•Bax∆2 aggregation is essential but not sufficient for cytotoxicity.•The C-terminus is critical for caspase 8-dependent cell death.•The C-terminal helix α9 structure is the key for caspase 8 recruitment.•The Bax family may have an intrinsic capability for caspase 8 activation.

Bax∆2 is a functional pro-apoptotic Bax isoform having alterations in its N-terminus, but sharing the rest of its sequence with Baxα. Bax∆2 is unable to target mitochondria due to the loss of helix α1. Instead, it forms cytosolic aggregates and activates caspase 8. However, the functional domain(s) responsible for BaxΔ2 behavior have remained elusive. Here we show that disruption of helix α1 makes Baxα mimic the behavior of Bax∆2. However, the other alterations in the Bax∆2 N-terminus have no significant impact on aggregation or cell death. We found that the hallmark BH3 domain is necessary but not sufficient for aggregation-mediated cell death. We also noted that the core region shared by Baxα and Bax∆2 is required for the formation of large aggregates, which is essential for BaxΔ2 cytotoxicity. However, aggregation by itself is unable to trigger cell death without the C-terminus. Interestingly, the C-terminal helical conformation, not its primary sequence, appears to be critical for caspase 8 recruitment and activation. As Bax∆2 shares core and C-terminal sequences with most Bax isoforms, our results not only reveal a structural basis for Bax∆2-induced cell death, but also imply an intrinsic potential for aggregate-mediated caspase 8-dependent cell death in other Bax family members.