S5D mutation of NLRP3 revealed its role in NLRP3 inflammasome complex using split-luciferase complementation assay

The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) is a critical cytosolic inflammasome sensor of exogenous and endogenous danger signals and is a highly attractive medicinal target against inflammatory diseases. The molecular mechanism leading to NLRP3 inflammasome activation through conformational changes of NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) proteins remains largely enigmatic. Here, we report several bioluminescent NLRP3 inflammasome reporters based on split-luciferase complementation assay to monitor structural and molecular changes of NLRP3 inflammasome. The designed split-luciferase NLRP3 reporter showed that NLRP3 molecules are in close spatial proximity in the resting state and undergo dynamic rearrangements upon nigericin stimulation. The reporter was responsive to the probable structural changes caused by MCC950 as a specific small-molecule inhibitor of NLRP3 oligomerization. Using these reporters, our findings indicate that while serine 5 of NLRP3 plays a crucial role in NLRP3 inflammasome regulation, it does not appear to be essential for NLRP3 oligomerization. Instead, it appears to be involved in the interaction of NLRP3 and ASC. To further investigate the molecular mechanisms underlying the impact of the serine 5 on NLRP3 inflammasome function, we conducted protein docking simulations. The molecular modeling revealed that the mutation of serine 5 to aspartate indirectly disrupts the interaction between aspartate 31 of NLRP3 and arginine 5 of ASC within the NLRP3-ASC interface. These findings demonstrate the utility of the reporters for monitoring interactions within the NLRP3 inflammasome, providing a platform for measuring dynamic changes of NLRP3 inflammasome and elucidating the mode of action of therapeutic agents.