Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

Sborgi, Lorenzo; Ravotti, Francesco; Dandey, Venkata P.; Dick, Mathias S.; Mazur, Adam; Reckel, Sina; Chami, Mohamed; Scherer, Sebastian; Huber, Matthias; Böckmann, Anja; Egelman, Edward H.; Stahlberg, Henning; Brož, Petr; Meier, Beat H.; Hiller, Sebastian

doi:10.1073/pnas.1507579112

Cited by 135 publications

(130 citation statements)

References 44 publications

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“…Speck formation is observed irrespective of which receptor is activated and ASC specks were even found to be released into the extracellular space, where they enhance inflammatory responses 77,78 . ASC specks appear to be formed by filaments of ASC 77 , which is consistent with cryo-electron microscopy and solid-state nuclear magnetic resonance analysis studies that show that human and mouse ASCs oligo merize through their PYDs into long helical filaments 79,80 . The ASC CARD is exposed on the surface of these filaments 80 and acts as a recruitment point for pro-caspase 1.…”

Section: Apoptosomesupporting

confidence: 79%

Inflammasomes: mechanism of assembly, regulation and signalling

Brož

Dixit²

2016

Nat Rev Immunol

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2,443

2,168

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Inflammasomes are multiprotein signalling platforms that control the inflammatory response and coordinate antimicrobial host defences. They are assembled by pattern-recognition receptors following the detection of pathogenic microorganisms and danger signals in the cytosol of host cells, and they activate inflammatory caspases to produce cytokines and to induce pyroptotic cell death. The clinical importance of inflammasomes reaches beyond infectious disease, as dysregulated inflammasome activity is associated with numerous hereditary and acquired inflammatory disorders. In this Review, we discuss the recent developments in inflammasome research with a focus on the molecular mechanisms that govern inflammasome assembly, signalling and regulation.

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Section: Apoptosomesupporting

confidence: 79%

Inflammasomes: mechanism of assembly, regulation and signalling

Brož

Dixit²

2016

Nat Rev Immunol

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2,443

2,168

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“…Similarly to what was previously found in PYD fibrils (18,39), the topology of the ring (Fig. 15B) makes the CARD domains of ASC molecules accessible for further interaction given the flexibility of the linker between ASC PYD and CARD domains (29).…”

Section: Figure 11 Nlrp3 Pyd Interacts With Asc Pyd Through Similar supporting

confidence: 73%

“…Unfortunately, this mutation has been shown to diminish the binding capabilities of the protein (see below). Remarkably, recent advances in cryo-EM imaging and solid-state NMR enabled the reconstitution at quasi-atomic resolution of ASC PYD inside a fibril, providing novel structural details into the homo-oligomerization mechanism of ASC (18,39). However, these groundbreaking structural reconstitutions only provide detailed characterization on mature fibrils, leaving the initial stages of aggregation still unrevealed.…”

Section: Asc Pyd Self-associates Through Two Opposing Bindingmentioning

confidence: 99%

“…Despite the abundant structural information reported for PYD domains (the three-dimensional structures of 10 different PYD domains have been deposited in the PDB) (6,17,29,(32)(33)(34)(35)(36)(37)(38), our understanding on the ASC⅐NLRP3 PYD homotypic interaction is very limited. Several models for this interaction have been proposed based on the NLRP3 crystallographic structure and mutational data (4,16), in addition to the recent semi-atomic structure characterization of ASC PYD mature fibril assemblies by cryo-EM and solid-state NMR (18,39).…”

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confidence: 99%

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ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces

Oroz

Barrera-Vilarmau

Alfonso³

et al. 2016

Journal of Biological Chemistry

100

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Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC⅐ASC and ASC⅐NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.Proteins of the death domain superfamily mediate in apoptotic, innate immunity, and inflammatory responses mainly through homotypic interactions (1). Specifically, they are responsible for the assembly of large signaling complexes in which kinases and caspases are activated (1-4). Despite the lack of sequence convergence within the superfamily, its members are characterized by adopting the so-called death fold, consisting of a globular fold with six ␣-helices arranged in a greek-key topology (1, 3). However, some structural differences between the subfamilies are observed within this common fold, including different charge distributions throughout the protein surface, diverse locations of hydrophobic patches, and the different lengths of certain ␣-helices. These differences are thought to lead to homotypic binding specificity (5), albeit certain heterotypic interactions are also known with proposed inhibitory roles in apoptotic and inflammatory pathways (1, 6).Detailed structural information on complexes formed by several members of the death domain superfamily is available. Some examples are the death domain interactions in the PIDDosome (7), the MyDDosome (8), the Fas⅐FADD (9, 10), and the Pelle⅐Tube death domain complexes (11), among others...

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“…Solid state NMR and cryoEM were also combined to solve the mammalian ASC inflammasome structure [35]. Solid-state NMR identified the mouse protein domain involved in filament assembly.…”

Section: Structure Determination By Nmr and Cryoem Of Self-assembled mentioning

confidence: 99%

Structures of biomolecular complexes by combination of NMR and cryoEM methods

Cuniasse

Tavares

Orlova

et al. 2017

Current Opinion in Structural Biology

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CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid-or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics. Modeling based on solid-state NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR became an established hybrid approach in structural biology that significantly contributes to our understanding of functional mechanisms in supramolecular assemblies. Highlights CryoEM combined with NMR provided atomic models of biocomplexes.  NMR structures together with X-ray structures enabled interpretation of cryoEM maps.  Solid-state NMR and cryoEM resolved structures of helical filaments and fibrils.  These approaches validated and improved models built de novo using cryoEM maps.  NMR structures were compared with cryoEM models to uncover assembly mechanisms.2

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Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

Cited by 135 publications

References 44 publications

Inflammasomes: mechanism of assembly, regulation and signalling

Inflammasomes: mechanism of assembly, regulation and signalling

ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces

Structures of biomolecular complexes by combination of NMR and cryoEM methods

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