title: Formation and structure of a NAIP5-NLRC4 inflammasome induced by direct interactions with conserved N- and C-terminal regions of flagellin.
abstract: The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome.
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Els F
authors.initials: EF
authors.lastname: Halff
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Diebolder
authors.forename: Marian
authors.initials: M
authors.lastname: Versteeg
authors.forename: Arie
authors.initials: A
authors.lastname: Schouten
authors.forename: T Harma C
authors.initials: TH
authors.lastname: Brondijk
authors.forename: Eric G
authors.initials: EG
authors.lastname: Huizinga
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry*; D053468:CARD Signaling Adaptor Proteins / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D020170:Caspase 1 / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005408:Flagellin / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D051616:Neuronal Apoptosis-Inhibitory Protein / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D058847:Inflammasomes; C512299:Ipaf protein, mouse; D008024:Ligands; C496800:NAIP protein, human; C435692:NLRC4 protein, human; C496806:Naip5 protein, mouse; D051616:Neuronal Apoptosis-Inhibitory Protein; D005408:Flagellin; D020170:Caspase 1