Following invasion by disease-causing biological entities, before a threat-specific response is mounted by the adaptive immune system, the innate immune system initiates a campaign to restrict the pathogen. In animals and plants, members of the Nucleotide-binding domain, Leucine-rich repeat-containing (NLR) superfamily of proteins are sentinels of the innate immune system that detect a wide array of pathogens’ molecular signatures.

NAIP5 (NLR family, apoptosis inhibitory protein 5), for example, is activated by binding the bacterial protein flagellin—a component of the flagellum many bacteria use for locomotion. After binding, the NAIP5–flagellin complex associates with multiple NLRC4 (NLR family, CARD [Caspase Activation and Recruitment Domain]-containing 4) protomers, forming an inflammasome that activates the protease Caspase-1 by juxtaposing the protease’s CARDs. The active Caspase-1 sparks a cascade that results in pyroptosis, a programmed form of cell death that summons an immune response and causes inflammation. Another such protein, NLRP1 (NLR family, pyrin domain containing 1), also forms inflammasomes that, when activated by cleavage by anthrax lethal factor, activate Caspase-1.

I used cryo-electron microscopy (cryo-EM) to determine the structure of the NAIP5–NLRC4 inflammasome. Analysis of the structure of the NAIP5-NLRC4 inflammasome revealed how the bacterial ligand flagellin is detected and how the complex assembles and uncovered a possible mechanism by which NLRs restrict pathogen evasion of detection by the innate immune system. Structural investigation of the NLRP1 inflammasome is at an early—but promising—stage. In this thesis, I also describe the new methods for preparing cryo-EM samples that made this work possible and may be useful for cryo-EM studies of other macromolecular complexes.