title: Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy.
abstract: Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland;
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Sborgi
authors.affiliation: Physical Chemistry, Swiss Federal Institute of Technology in Zurich, 8093 Zurich, Switzerland;
authors.forename: Francesco
authors.initials: F
authors.lastname: Ravotti
authors.affiliation: Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, 4058 Basel, Switzerland;
authors.forename: Venkata P
authors.initials: VP
authors.lastname: Dandey
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland;
authors.forename: Mathias S
authors.initials: MS
authors.lastname: Dick
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland; Research IT, Biozentrum, University of Basel, 4056 Basel, Switzerland;
authors.forename: Adam
authors.initials: A
authors.lastname: Mazur
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland;
authors.forename: Sina
authors.initials: S
authors.lastname: Reckel
authors.affiliation: Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, 4058 Basel, Switzerland;
authors.forename: Mohamed
authors.identifier: http://orcid.org/0000-0002-8733-055X
authors.initials: M
authors.lastname: Chami
authors.affiliation: Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, 4058 Basel, Switzerland;
authors.forename: Sebastian
authors.initials: S
authors.lastname: Scherer
authors.affiliation: Physical Chemistry, Swiss Federal Institute of Technology in Zurich, 8093 Zurich, Switzerland;
authors.forename: Matthias
authors.initials: M
authors.lastname: Huber
authors.affiliation: Institute for the Biology and Chemistry of Proteins, 69367 Lyon, France;
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908.
authors.forename: Edward H
authors.initials: EH
authors.lastname: Egelman
authors.affiliation: Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, 4058 Basel, Switzerland;
authors.forename: Henning
authors.initials: H
authors.lastname: Stahlberg
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland; petr.broz@unibas.ch beme@ethz.ch sebastian.hiller@unibas.ch.
authors.forename: Petr
authors.initials: P
authors.lastname: Broz
authors.affiliation: Physical Chemistry, Swiss Federal Institute of Technology in Zurich, 8093 Zurich, Switzerland; petr.broz@unibas.ch beme@ethz.ch sebastian.hiller@unibas.ch.
authors.forename: Beat H
authors.initials: BH
authors.lastname: Meier
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland; petr.broz@unibas.ch beme@ethz.ch sebastian.hiller@unibas.ch.
authors.forename: Sebastian
authors.identifier: http://orcid.org/0000-0002-6709-4684
authors.initials: S
authors.lastname: Hiller
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D053468:CARD Signaling Adaptor Proteins; D003001:Cloning, Molecular; D020285:Cryoelectron Microscopy; D058847:Inflammasomes / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008958:Models, Molecular; D011487:Protein Conformation
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D058847:Inflammasomes; C498534:Pycard protein, mouse
keywords: ASC speck; inflammation; innate immune response; protein filament; protein structure