Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2PYD filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASCPYD filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently demonstrate that individual filaments assemble bidirectionally. By contrast, the recognition between AIM2 and ASC requires at least one to be oligomeric and occurs in a head-to-tail manner. Using in silico mutagenesis as a guide, we also identify specific axial and lateral interfaces that dictate the recognition and distinction between AIM2 and ASC filaments. Together, the results here provide a robust framework for delineating the signaling specificity and order of inflammasomes.

Edited by Ruma BanerjeeInflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The NOD-like receptor (NLR) family apoptosis inhibitory protein (NAIP)⅐NLR family caspase-recruiting domain (CARD) domain-containing 4 (NLRC4) inflammasome recognizes intracellular bacteria and induces the polymerization of the caspase-1 protease, which in turn executes maturation of interleukin-1␤ (IL-1␤) and pyroptosis. Several high-resolution structures of the fully assembled NAIP⅐NLRC4 complex are available, but these structures do not resolve the architecture of the CARD filament in atomic detail. Here, we present the cryo-EM structure of the filament assembled by the CARD of human NLRC4 (NLRC4 CARD ) at 3.4 Å resolution. The structure revealed that the helical architecture of the NLRC4 CARD filament is essentially identical to that of the downstream filament assembled by the CARD of caspase-1 (casp1 CARD ), but deviates from the split washer-like assembly of the NAIP⅐NLRC4 oligomer. Our results suggest that architectural complementarity is a major driver for the recognition between upstream and downstream CARD assemblies in inflammasomes. Furthermore, a Monte Carlo simulation of the NLRC4 CARD filament assembly rationalized why an (un)decameric NLRC4 oligomer is optimal for assembling the helical base of the NLRC4 CARD filament. Together, our results explain how symmetric and asymmetric supramolecular assemblies enable high-fidelity signaling in inflammasomes.

Inflammasomes are filamentous signaling platforms essential for host defense against various intracellular calamities such as pathogen invasion and genotoxic stresses. However, dysregulated inflammasomes cause an array of human diseases including autoinflammatory disorders and cancer. It was recently identified that endogenous pyrin-only-proteins (POPs) regulate inflammasomes by directly inhibiting their filament assembly. Here, by combining Rosetta in silico, in vitro, and in cellulo methods, we investigate the target specificity and inhibition mechanisms of POPs. In contrast to a previous report, we find that POP1 is a poor inhibitor of the central inflammasome adaptor ASC. Instead, POP1 inhibits the assembly of upstream receptor PYD filaments such as those of AIM2, IFI16, NLRP3, and NLRP6. Moreover, not only does POP2 directly suppress the nucleation of ASC, but it can also inhibit the elongation of receptor filaments. In addition to inhibiting the elongation of AIM2 and NLRP6 filaments, POP3 potently suppresses the nucleation of ASC. Our Rosetta analyses and biochemical experiments consistently suggest that a combination of favorable and unfavorable interactions between POPs and PYDs is necessary for effective recognition and inhibition. Together, we reveal the intrinsic target redundancy of POPs and their inhibitory mechanisms.