Inga Hochheiser

Conformational transitions in NLRP3 inflammasome formation

Innate immune cells recognise invading pathogens via germline-encoded pattern recognition receptors (PRRs). NOD-like receptor protein 3 (NLRP3) is a cytosolic PRR that senses a diverse set of stimuli, including pathogen-derived molecular patterns or endogenous danger signals. Upon activation, NLRP3 forms a multiprotein signalling platform called the inflammasome, which induces the production of proinflammatory cytokines and pyroptotic cell death. NLRP3 is the clinically most implicated inflammasome protein with its overdue activation being involved in a broad range of chronic inflammatory diseases, including atherosclerosis, diabetes and Alzheimer’s disease. Although NLRP3 is a highly relevant drug target, the mechanisms leading to NLRP3 activation and the functioning of antagonistic small molecules remain poorly understood. We investigated the structures of different NLRP3 functional states to delineate NLRP3 inflammasome formation. We found that inactive, ADP-bound NLRP3 forms a spherical decamer with a size of 1.2 megadaltons. Binding of the NLRP3-specific antagonist CRID3 at the backside of the NLRP3 nucleotide binding domain locks NLRP3 in its inactive conformation and prevents downstream activation. Active NLRP3 via homotypic pyrin domain (PYD) interactions with its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) oligomerizes into helical, filamentous assemblies. We determined the structure of the human NLRP3-PYD filament, showing that NLRP3- and ASC-PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing a continuous transition between NLRP3 as the nucleation seed and ASC as the elongator. Analysing the NLRP3 to ASC filament transition by cryo-EM, we show that NLRP3-induced inflammatory responses exhibit a particular directionality.