Biochemical and structural analysis of the NLRP3 inflammasome

• Applicant: Professor Dr. Matthias Geyer
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504829198

Mammals possess a very effective and complex immune system to counteract pathogens threatening their physical health. Inflammasomes are cytosolic multi-protein complexes that form in response to a wide range of pathogens, tissue damage and other harmful stimuli in order to trigger innate immune responses. Members of the family of NOD-like receptors (NLRs) sense these pathogen and danger associated molecular patterns, which leads to their activation and oligomerization. NLRP3 is possibly the most prominent inflammasomal receptor protein as it senses a broad variety of stimuli and is involved in multiple autoinflammatory diseases. Yet, the structural basis of inflammasome function and the molecular transition from an autoinhibited state into a fully activated state is only poorly understood. In preliminary work we have purified human full length, wild type NLRP3 protein from baculo virus infected insect cells to homogeneity. Unexpectedly, the protein elutes at a size of 1.15 MDa indicating a large oligomeric assembly. Using single particle cryo-EM, we determined the structure of NLRP3 in the inactive state bound to the cytokine release inhibitory drug CRID3 at 3.9 Ang resolution. ADP-bound NLRP3 is a decamer composed of homodimers of intertwined LRR domains that assemble back-to-back as pentamers. The NACHT domain is located at the apical axis of this spherical structure. We now want to address two major aims: (1) The cryo-EM structure determination of the oligomeric NLRP3 complex in an active state, and (2) the functional characterization of the different conformational states of NLRP3 by biochemical means. Besides state of the art cryo-EM imaging and structure reconstruction methods, the research aims shall be achieved in eight work packages, which includes conversion of the NLRP3 decamer into an active state, implications of the kinase NEK7 on the conformational states of NLRP3, the set-up of an NLRP3-stimulated ASC filament seeding assay, the structural and functional characterization of a membrane associated NLRP3 species, analysis of ATP hydrolysis, characterization of disease mutations and autoinhibition, the interaction with model membranes, and the monitoring of conformational changes upon in vitro phosphorylation of NLRP3. These analyses will help elucidating the structure–function relationship in NLRP3 and provide insights into the conformational states of this medicinally important inflammasome.

• DFG Programme: Research Grants