Cryo-EM structure of the NLRC4CARD filament provides insights into how symmetric and asymmetric supramolecular structures drive inflammasome assembly

Abstract

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.

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

“…S5d-e). The final polished structure closely agrees with previously published findings 20,34 . Interestingly, unlike NLRP1-CARD and CARD8-CARD, the strong density bridge in the Type II interface is absent within NLRC4-CARD filaments 13 ( Fig.…”

“…4a-b to Fig. 3c-d), which had not been highlighted by previous analyses 20,27,[29][30][31][32][33][34][35] . In ASC-CARD filaments, this bridge consists of two residues Gln185 and Tyr187, which extend from the Type IIb surface and interact with Ala168 and Asn170 on the Type IIa surface ( Fig.…”

Novel mode of filament formation in UPA-promoted CARD8 and NLRP1 Inflammasomes

“…Like other CARD filament structures 4,7,33,34,36,37 , the CARD8 CARD filament also possesses a one-start helical symmetry, with a refined left-handed rotation of 99.1° and an axial rise of 5.2 Å per subunit (~3.6 subunits per helical turn). The filament has a dimeter of ~80 Å with a central hole of ~10 Å ( Fig.…”

“…4a). The NLRP1 CARD filament has onestart helical symmetry, with a left-handed rotation of 100.8° and an axial rise of 5.1 Å per subunit (~3.6 subunits per helical turn), similar to other CARD filament structures 4,7,33,34,36,37 . Strikingly however, unlike any other CARD filament structures known to date, the NLRP1 CARD filament is composed of NLRP1 CARD dimers, rather than monomers ( Fig.…”

“…While UPA-CARD formed thick filaments, CARD alone only formed thin filaments that are characteristic of the inner CARD ( Fig. 4e) and of other published CARD filaments 4,7,33,34,36,37 . These data suggest that UPA dimerization or oligomerization promotes NLRP1 CARD dimerization, as well as NLRP1 helical filament formation, strengthening the role of UPA in the NLRP1 signalling paradigm.…”