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

Abstract

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIIND UPA -CARD) of NLRP1 and CARD8 self-oligomerize to trigger the assembly of distinct inflammasome complexes. Recombinant FIIND UPA -CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIIND UPA rings.Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIIND UPA , which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 Å, which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

“…In contrast to free UPA 28,29 , the FIIND was monomeric in solution (Extended Data Fig. 8g), which suggests that the ZU5 domain inhibits UPA dimerization or oligomerization.…”

Structural mechanism of CARD8 regulation by DPP9

“…Despite maintaining FIIND autoprocessing, interface III mutations completely abolished inflammasome activity, even in the presence of the activating ligand VbP (Figure 3D). We reasoned that the observed UPA-UPA interface might be preserved on the inflammasome filament, as the UPA itself promotes CARD oligomerization and inflammasome activity (Hollingsworth et al, 2020a; Qin et al, 2020). Additionally, NLRP1 interface III mutations abolished inflammasome signaling in cells (Hollingsworth et al, 2020b) and disrupted filament formation in vitro (Huang et al, 2020).…”

“…However, it should be noted that our studies suggest that CARD8 and NLRP1 may have also both evolved to sense a single endogenous danger signal triggered by DPP8/9 inhibition. Another important difference between CARD8 and NLRP1 is the structure of assembled inflammasome: while CARD8 can only directly recruit caspase-1 and activate rapid pyroptosis without causing cytokine maturation, NLRP1 recruits caspase-1 via ASC to elicit an inflammatory signaling cascade with cytokine secretion and pyroptotic cell death (Ball et al, 2020; Hollingsworth et al, 2020a; Qin et al, 2020). In this context, it is possible that NLRP1 activation is more pro-inflammatory, as exemplified by the association of NLRP1 hyperactivation with a series of severe skin pro-inflammatory diseases (Grandemange et al, 2017; Jin et al, 2007; Levandowski et al, 2013; Zhong et al, 2016; Zhong et al, 2018).…”

“…The cryo-EM maps were first fit with the crystal structure of DPP9 dimer (PDB ID: 6EOQ) (Ross et al, 2018). A homology model of CARD8-FIIND was generated with Schrodinger Prime (Jacobson et al, 2002) using the structure of NLRP1-FIIND (Hollingsworth et al, 2020a; Qin et al, 2020) as template. Manual adjustment and de novo building of missing segments, rigid-body fitting, flexible fitting, and segment-based real-space refinement were performed in distinct parts of the initial model to fit in the density in Coot (Emsley et al, 2010), with help of UCSF-Chimera (Goddard et al, 2007) and real-space refinement in Phenix (Klaholz, 2019).…”

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

“…We hypothesize that the same UPA-UPA interaction surface as observed in the 2:1 NLRP1-DPP9 complex is utilized in the higher order UPA-CARD filament. Indeed, negative staining EM showed that the wild-type UPA-CARD of hNLRP1 formed filamentous structures 25,26 (Fig. 5b), but not the UPA-UPA interface mutants, UPA-CARD P1278E and UPA-CARD L1281E (Fig.…”