The dynamic and heterogeneous structure of the non-canonical inflammasome

Inflammasomes are high-molecular-weight complexes that play integral roles in the innate immune system, triggering an inflammatory cascade to protect against cellular stresses such as pathogenic bacteria. Both canonical and non-canonical inflammasomes have been described in the literature and detailed structural studies of many components of the more complex and larger canonical versions have been reported. In contrast, corresponding structures of the non-canonical inflammasome have not emerged even though it consists of only two components: lipopolysaccharide (LPS) from Gram-negative bacteria and one of caspase-4 or caspase-5 in humans or caspase-11 in mice. Here, we determine the stoichiometry of the non-canonical inflammasome showing that it is heterogeneous, comprised of three major complexes with different numbers of LPS and caspase molecules. NMR spectroscopy studies of the N-terminal caspase activation and recruitment domain (CARD) of caspase-11, which binds LPS, establish that it is largely unstructured in the absence of lipid, with pervasive dynamics on the µs-ms timescale. Formation of this complex increases the α-helical content of the CARD, but the dynamics persist, multiple conformers are formed, and tertiary contacts are transient. The NMR results presented establish that the protease domain of caspase-11 is monomeric in isolation. As proteolysis is linked with dimerization, the protease domains are inactive in this state, but upon formation of the non-canonical inflammasome, dimerization occurs, priming the complex for rapid processing of substrates. Our study highlights the utility of NMR for studies of heterogeneous systems that are recalcitrant to crystallographic or cryo-EM analyses.