Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation

Abstract

Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)–like receptors with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) mediate oligomerization into filamentous assemblies. We describe the cryo–electron microscopy (cryo-EM) structure of the human NLRP3 PYD filament and identify a pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends designated as A- and B-ends. Coupling a titration polymerization assay to cryo-EM, we demonstrate that ASC adaptor protein elongation on NLRP3 PYD nucleation seeds is unidirectional, associating exclusively to the B-end of the filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing a continuous transition between NLRP3 and ASC. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3, ASC, and Caspase-1 proteins.

Filament assembly underpins the double-stranded DNA specificity of AIM2-like receptors

“…To further investigate the apparent lack of interactions between ASC PYD and AIM2 polymers assembled on nucleic acids other than dsDNA, we imaged the upstream and downstream signaling partners together via nsEM. Here, we observed ASC PYD filament (∼9 nm diameter; ( 44 )) apparently growing off from one end of AIM2 FL •dsDNA filaments (∼25 nm diameter; ( 36 , 37 )) (Figure 3C ), which is consistent with the notion that ASC assembles from one specific end of receptor filaments ( 37 , 41 , 45 ). Interestingly, ASC PYD also appeared to make a directional contact with the AIM2 FL •poly(IC) complexes (Figure 3C ).…”

Design Principles for Inflammasome Inhibition by Pyrin-Only-Proteins

“…The InterfaceAnalyzer script in Rosetta was used to determine the interaction energy at individual interfaces of the honeycomb (Matyszewski et al, 2021). We used the cryo-EM structures of ASC PYD (PDB: 3j63; (Lu et al, 2014)), AIM2 PYD (PDB: 7k3r; (Matyszewski et al, 2021)), NLRP3 PYD (PDB: 7pdz; (Hochheiser, Behrmann, et al, 2022)) and NLRP6 PYD (PDB: 6ncv; (Shen et al, 2019)) filaments to generate corresponding honeycombs.…”

“…For instance, consistent with our Rosetta analyses showing that homotypic PYD•PYD interactions are preferred in all cases, excess POPs were required to suppress the assembly of inflammasome PYDs (e.g., Figures 2E, 3E ). PYD filaments are highly organized helical structures-all six interfaces from each protomer are necessary to assemble into intact, functional PYD filaments (Hochheiser, Behrmann, et al, 2022;Lu et al, 2014;Matyszewski et al, 2021;Shen et al, 2019). We postulate that even weakly favorable interactions at one surface (e.g., Type 3a between POP2/3 and NLRP6 PYD ) would be sufficient for POPs associate with their targets and interfere with filament assembly via any unfavorable interactions at the other five available surfaces.…”

“…We then calculated Rosetta interface energies (∆Gs) for each PYD filament (e.g., Figure 1A, left), and also determined the ∆Gs for POP•PYD interactions by replacing the center protomer with each POP (e.g., Figure 1A, three honeycombs on the right). Of note, we decided to conduct our in silico and subsequent biochemical experiments on tractable inflammasome components with well-known biological significances such as ASC, AIM2, IFI16, NLRP3, and NLRP6 (Broz & Dixit, 2016;Hochheiser, Behrmann, et al, 2022;Kerur et al, 2011;Lu et al, 2014;Matyszewski et al, 2018;Matyszewski et al, 2021;Morrone et al, 2015;Morrone et al, 2014;Shen et al, 2019).…”

“…Inflammasome filaments are highly ordered supra-structures that entail at least two distinct steps for assembly: rate-limiting nucleation followed by elongation (Kagan et al, 2014;Lu et al, 2014;Matyszewski et al, 2018). Moreover, although the PYDs of upstream receptors do not display significant a.a. sequence homologies (Kagan et al, 2014;Lu et al, 2014;, they all assemble into structurally congruent helical filaments and signal through the common ASC adaptor, suggesting a degenerate-code-like recognition mechanism (Hochheiser, Behrmann, et al, 2022;Kagan et al, 2014;Lu et al, 2014;Matyszewski et al, 2021;Shen et al, 2019). At present, little is known about how POPs selectively target and regulate the assembly of such diverse yet homologous supramolecular structures.…”