LPS-induced structural reorganization and polymerization drive noncanonical inflammasome activation.

The noncanonical inflammasome, mediated by murine caspase-11 and its human orthologs caspase-4 and caspase-5, detects intracellular lipopolysaccharide (LPS) and triggers pyroptotic cell death. Upon LPS binding through their amino-terminal caspase activation and recruitment domains (CARDs), these inflammatory caspases oligomerize and activate. However, how LPS binding drives caspase-4/11 activation remains unclear. Here, we show that caspase-4/11 CARDs are intrinsically unstructured in their resting state and adopt an α-helical conformation upon LPS engagement. This structural rearrangement promotes CARD oligomerization, with electron-capture charge reduction-coupled native mass spectrometry and other techniques illustrating that hexa-acylated LPS induces the formation of large oligomers composed of eight or more protomers, whereas underacylated lipids trigger smaller assemblies. Using hydrogen-deuterium exchange mass spectrometry and cross-linking mass spectrometry, we identified a hydrophobic cleft critical for LPS binding and potential intersubunit interfaces that facilitate oligomerization. These findings uncover key structural features underlying LPS recognition and activation of the noncanonical inflammasome, providing mechanistic insight into an essential arm of the innate immune response.