Cryo-EM structures of ASC and NLRC4 CARD filamentsreveal a unified mechanism of nucleation andactivation of caspase-1Yang Lia,b,1,2, Tian-Min Fua,b,1,3, Alvin Lua,b,4, Kristen Witta,b, Jianbin Ruana,b, Chen Shena,b, and Hao Wua,b,3

aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and bProgram in Cellular and MolecularMedicine, Boston Children’s Hospital, Boston, MA 02115

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2015.

Contributed by Hao Wu, August 17, 2018 (sent for review June 19, 2018; reviewed by Tsan Sam Xiao and Rui Zhang)

Canonical inflammasomes are cytosolic supramolecular complexesthat activate caspase-1 upon sensing extrinsic microbial invasionsand intrinsic sterile stress signals. During inflammasome assembly,adaptor proteins ASC and NLRC4 recruit caspase-1 throughhomotypic caspase recruitment domain (CARD) interactions, lead-ing to caspase-1 dimerization and activation. Activated caspase-1processes proinflammatory cytokines and Gasdermin D to inducecytokine maturation and pyroptotic cell death. Here, we presentcryo-electron microscopy (cryo-EM) structures of NLRC4 CARD andASC CARD filaments mediated by conserved three types of asym-metric interactions (types I, II, and III). We find that the CARDs ofthese two adaptor proteins share a similar assembly pattern, whichmatches that of the caspase-1 CARD filament whose structure wedefined previously. These data indicate a unified mechanism fordownstream caspase-1 recruitment through CARD–CARD interac-tions by both adaptors. Using structure modeling, we further showthat full-length NLRC4 assembles via two separate symmetries at itsCARD and its nucleotide-binding domain (NBD), respectively.

ASC | NLRC4 | inflammasome | caspase-1 | CARD

As the first line of defense, the innate immune system em-ploys a variety of pattern recognition receptors (PRRs) to

detect pathogen-associated molecular patterns (PAMPs) anddamage-associated molecular patterns (DAMPs) (1–3). So far, atleast five families of PRRs have been characterized, includingToll-like receptors (TLRs), RIG-I–like receptors (RLRs), C-typelectin receptors (CLRs), AIM2-like receptors (ALRs), and nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containingproteins (NLRs) (4). Of these, upon ligand stimulation, ALRs andsome NLRs have been shown to form oligomeric supramolecularcomplexes known as canonical inflammasomes, which also containadaptor proteins and caspase-1 (Casp-1) (2, 3) (Fig. 1A). Exam-ples of canonical inflammasomes include, but are not limited to,the AIM2 inflammasome, the NLRP1 inflammasome, the NLRP3inflammasome, and the NAIP inflammasomes (2).Different inflammasomes are responsible for recognition of,

and activation by, different ligands. For example, AIM2 recog-nizes double-stranded DNA in the cytosol (5–7); NLRP3 re-sponds to K+ efflux that is in turn induced by multiple stimuli,such as extracellular ATP, uric acid crystals, and the bacterialtoxin nigericin (8); and NAIP proteins detect flagellin and com-ponent proteins of the bacterial type III secretion system (9–12).Ligand binding activates these proteins to recruit adaptor pro-teins, such as ASC and NLRC4, which subsequently engage thedownstream effector caspase-1. Most inflammasomes use theASC adaptor, which possesses an N-terminal Pyrin domain (PYD)and a C-terminal caspase recruitment domain (CARD) (13) (Fig.1A). The N-terminal PYD interacts with the PYD of the upstreamsensors, and the C-terminal CARD recruits caspase-1 via homo-typic CARD–CARD interactions (13, 14). In contrast, the NLRC4adaptor exists only in NAIP inflammasomes, bridging an NAIP

via its NBD and LRR and caspase-1 via its CARD upon ligandstimulation (15–19) (Fig. 1A). As the universal effector of canon-ical inflammasomes, caspase-1 is recruited and polymerized throughits CARD to form filamentous structures, bringing the caspasecatalytic domains into proximity and leading to its dimerizationand activation (14). Activated caspase-1 processes cytokines pro–IL-1β and pro–IL-18 to their mature forms to elicit inflammatoryresponses and cleaves Gasdermin D to form pores that releasethe cytokines and cause pyroptotic cell death (20–25).As the first CARD filament structure in inflammasomes,

Casp-1CARD revealed the molecular mechanism of its self-assembly,as well as regulation by CARD-only proteins INCA and ICERBERG(14). However, how the upstream adaptors nucleate Casp-1CARD

filament assembly still remains elusive. By visualizing the structures

Significance

Inflammasomes are cytosolic protein complexes that detect thepresence of pathogens and damages to elicit immune responses,and dysregulation in inflammasome signaling is associated withmany human diseases. As the unified downstream effector ofcanonical inflammasomes, caspase-1 is recruited though CARD–CARD interactions with the adaptor proteins ASC or NLRC4. Wehave determined the cryo-EM structures of ASC CARD andNLRC4 CARD filaments. Using multidisciplinary methods, wereveal a common mechanism of caspase-1 CARD nucleation,assembly, and activation by equivalent assembly patterns inASC and NLRC4. Collectively, our data provide insights intoinflammasome assembly and activation and afford structuralplatforms for modulating these CARD–CARD interactions inpotential therapeutic applications.

Author contributions: Y.L., T.-M.F., and H.W. designed research; Y.L., T.-M.F., A.L., K.W.,J.R., and C.S. performed research; Y.L., T.-M.F., A.L., and H.W. analyzed data; and Y.L.,T.-M.F., and H.W. wrote the paper.

Reviewers: T.S.X., Case Western Reserve University; and R.Z., Washington University.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.wwpdb.org [PDB ID codes 6DRN (ASCCARD filaments) and 6DRP (NLRC4CARD fila-ments)], and the cryo-EM reconstructions have been deposited in the EM Data Bank,www.emdatabank.org [ID codes EMD-8902 (ASCCARD filaments) and EMD-8903(NLRC4CARD filaments)].1Y.L. and T.-M.F. contributed equally to this study.2Present address: Department of Biophysics, University of Texas Southwestern MedicalCenter, Dallas, TX 75390.

3To whom correspondence may be addressed. Email: wu@crystal.harvard.edu or Tianmin.Fu@childrens.harvard.edu.

4Present address: Department of Biological Sciences, Ribon Therapeutics, Inc., Lexington,MA 02421.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810524115/-/DCSupplemental.

Published online October 2, 2018.

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of ASCCARD and NLRC4CARD filaments using cryo-EM, here,we reveal that ASC and NLRC4 adopt the same mechanism tonucleate the assembly and activation of caspase-1. The ASCCARD

and NLRC4CARD filament structures show assembly patternssimilar to that of Casp-1CARD, indicating that these adaptorstemplate the polymerization of Casp-1CARD. Further structuralanalyses and biochemical assays show that ASC and NLRC4utilize similar interfaces to recruit caspase-1 and confer a uni-directional polymerization of Casp-1CARD by charge and shapecomplementarity.

ResultsCryo-EM Reconstruction of the ASCCARD and NLRC4CARD FilamentStructures. Both ASCCARD and NLRC4CARD are capable of nu-cleating the assembly and activation of caspase-1 (Fig. 1A). Togain a mechanistic understanding of this process, we preparedASCCARD and NLRC4CARD filaments for cryo-EM study. Wefound that His-MBP-ASCCARD-SUMO was purified as monomersover a gel filtration column and ASCCARD-SUMO formed filamentsupon proteolytic removal of the His-MBP tag by the Tobacco EtchVirus (TEV) protease (Fig. 1 B and C). The averaged powerspectrum showed a similar diffraction pattern to that of Casp-1CARD

filaments (14). Based on possible indexing of the power spectrum,we adopted a calculated one-start helical symmetry with an azi-muthal angle of −100.60° and an axial rise of 5.10 Å per subunit.The iterative helical real-space reconstruction (IHRSR) method(26) was used to generate an intermediate map, starting from asolid cylinder as the initial model. This map was then used asan initial model in RELION (27) for 3D classification (SI Ap-pendix, Fig. S1) and refinement. The final volume containsmostly α-helices with the refined helical symmetry parameters of−100.58° rotation and 5.00 Å translation per subunit, respectively.Each subunit showed the typical six α-helices arranged in a Wshape, which is a common feature of the death domain super-family that includes the CARD (28) (Fig. 1D). The calculatedpower spectrum from the final volume corresponded well with theexperimental power spectrum (SI Appendix, Fig. S2A). The NMR

structure of ASCCARD (PDB ID code 2KN6) could be easilydocked into the EM density (Fig. 1D and SI Appendix, Table S1).The model was manually adjusted in Coot (29), followed byrefinement in Phenix (30). The amino acid sequence was un-ambiguously registered due to the clearly defined side chains.There is almost no density outside the filament, indicating that theC-terminal SUMO tag was largely disordered (Fig. 1D). Theresolution of this reconstruction was measured at 3.2 Å using thegold standard Fourier shell correlation (FSC) in RELION (SIAppendix, Fig. S3).In the case of the NLRC4CARD domain, we found that the

construct of GFP-NLRC4CARD directly forms filaments suitablefor structure determination (Fig. 1 B and E). As the cryo-EMimages showed, NLRC4CARD filaments are generally shorter andwider than ASCCARD filaments, likely due to the effect of thelarger GFP tag (Fig. 1 C and E). We employed a similar strategyas used for ASCCARD to determine the NLRC4CARD filamentstructure. The helical symmetry was first calculated from theaveraged power spectrum (SI Appendix, Fig. S2B) as −100.50° inazimuthal angle and 5.10 Å in axial rise per subunit, which wasrefined to −100.48° and 4.93 Å, respectively. The GFP tag islargely disordered in that the final reconstruction contains onlyweak noisy densities in the periphery. We also calculated thepower spectrum from the final volume, which matched well withthe experimental power spectrum (SI Appendix, Fig. S2B). Ahomology structure model of NLRC4CARD derived from theCasp-1CARD structure (PDB ID code 5FNA) (14) was readily fittedinto the cryo-EM density (SI Appendix, Table S2 and Fig. 1F).Similar to the case for ASCCARD, the obvious side chain densitiesof the NLRC4CARD map enabled manual model building in Coot(29), followed by refinement in Phenix (30). The resolution wasmeasured at 3.6 Å using gold standard FSC (SI Appendix, Fig. S4).

Structure of the ASCCARD Filament. The diameter of the ASCCARD

filament is ∼8 nm, with a central hole of less than 1 nm (Fig. 2A).The filament structure assembles through a left-handed one-starthelical symmetry, with about 3.6 subunits per turn (Fig. 2B). Like

A

D E F

B C

Fig. 1. Cryo-EM structure determination of ASCCARD and NLRC4CARD filaments. (A) A brief schematic for ASC and NLRC4 recruitment of caspase-1. (B) ASC andNLRC4 CARD constructs used for EM studies. (C) An electron micrograph of ASCCARD filaments. (D) Side view of EM reconstruction fitted with the ASCCARD

filament model with each subunit in a different color. One subunit is enlarged for closer view. (E) A micrograph of NLRC4CARD filaments. (F) Side view of EMreconstruction fitted with the NLRC4CARD filament model with each subunit in a different color. One subunit is enlarged for closer view.

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other filaments formed by members of the death domain su-perfamily, formation of the ASCCARD filament is mediated bythree types of asymmetric interactions, namely type I, type II,and type III interactions (28) (Fig. 2B). In the filament archi-tecture, the type III interaction is along the direction of the one-start helical strand while type I and type II interactions generateconnections between the adjacent turns of the helical strands(Fig. 2B). The type I interaction is mostly composed of charge–charge interactions between residues located on helix α2 of onemolecule and helices α1 and α4 of the partner molecule and isthe most extensive in surface area among the three types of in-teractions. Possible residues involved in this interaction includeR119, E130, D134, and R160 (Fig. 2C). These residues formseveral electrostatically complementary pairs. Unlike the type Iinteraction, the type II interaction in the ASCCARD filament ismainly contributed by hydrophobic residues, including W169 andY187 (Fig. 2C). The type III interaction is also dominated bycharge–charge interactions, with R160 of helix α4 and D143 andE144 of helix α3 forming charge complementary pairs at theinterface (Fig. 2C).

To validate the importance of the interfacial residues identi-fied by our structural analysis, we generated site-directed mutantson a construct of ASCCARD fused to GFP (GFP-ASCCARD). WTGFP-tagged ASCCARD primarily eluted at the void fraction on agel filtration column (Fig. 2D). In contrast, R119D, N128A/E130R,and D134K of type I mutations completely abolished filamentformation (Fig. 2D). The effectiveness of these charge-reversalmutations confirmed our observation that type I interaction isdominated by charge–charge interactions. In the case of type IIinteractions, W169G, Y187A, Y187K mutations almost com-pletely disrupted filament formation (Fig. 2D). Additionally,mutation of Y187 to L or H partially disrupted filament for-mation, further showing the hydrophobic interaction of the typeII interface (Fig. 2D). D143K/E144K and R160E of type III in-teractions almost completely disrupted filament formation (Fig.2D). We further examined mCherry-tagged ASCCARD mutantsusing confocal microscopy in HeLa cells. In line with our in vitrobiochemical data, WT ASCCARD formed filaments in cells whilemutations that proved to be disruptive in vitro also abolished fil-ament formation in cells (Fig. 2E). These mutagenesis studies

IIIa IIIb

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Gel Filtration Profile of ASCCARD Wild-type and Mutants mCherry BF Merge Hoechst

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Fig. 2. Structural analysis of the ASCCARD filament structure. (A) Surface representation of ASCCARD filament structure, side view and top view. (B) Schematicdiagram of the helical filament, with three neighboring subunits highlighted in green, magenta, and cyan. (C) Detailed type I, II, and III interfaces, re-spectively, of the ASCCARD filament structure. (D) Gel filtration profile of ASCCARD WT and mutants. Void fractions are from elution volumes 7 mL to 9 mL whileless aggregated fractions are from elution volumes 14 mL to 17 mL. (E) WT and ASCCARD mutants overexpressed in HeLa cells examined by confocal mi-croscopy. (Scale bar: 10 nm.)

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strongly support the correctness of our structural model andanalysis.

Structure of the NLRC4CARD Filament. The NLRC4CARD filamenthas a very similar architecture to the ASCCARD filament, with adiameter of ∼8 nm and an even smaller central hole (Figs. 2Aand 3A). Like the ASCCARD filament, each subunit of theNLRC4CARD filament interacts with its neighboring moleculesthrough three types of interactions, of which the type III in-teraction mediates the intrastrand contact and type I and type IIinteractions mediate interstrand contacts (Fig. 3 A and B). Thetype I interaction is composed of helix α2 of one molecule andhelices α1 and α4 of the other. Electrostatic complementaryresidues of R9, D25, D26, and R52 form charge–charge inter-actions at the interface (Fig. 3C). At the type II interface, K60/K61 and E47 interact with each other through charge comple-mentarity (Fig. 3C). Like the type I interface, the type III in-terface is mainly composed of hydrophilic residues, including thecharge pair consisting of K45 and E44 (Fig. 3C).To further validate the NLRC4CARD filament model, we

performed site-directed mutagenesis on the GFP tagged con-struct. While WT GFP-NLRC4CARD mainly eluted at the voidfraction, mutations of residues on type I and type III interfaces(R52E on type Ia, D25K on type Ib, and E36R on type IIIa)effectively abolished its aggregation ability (Fig. 3D). R9E ontype Ia and K60E/K61D double mutation on the type IIa in-terface led to partial disruption of filament formation (Fig. 3D).We also investigated eGFP-tagged NLRC4CARD mutants us-ing confocal microscopy in HeLa cells. Consistent with our

observation in vitro, WT NLRC4CARD formed filaments in cells,and surface mutants of all three types of interactions abolished orattenuated filament formation (Fig. 3E). Collectively, these mu-tagenesis data further support the helical assembly model of theNLRC4CARD filament.

The NLRC4CARD Filament Exists in the Full-Length NLRC4 Structure.Upon activation by a ligand-bound NAIP protein (18, 19),NLRC4 with the N-terminal CARD deleted forms mainly 11- to12-folded disk-shaped complexes with a central hole of ∼8 nm indiameter and an outer diameter of ∼30 nm (16, 17). The size ofthe hole is compatible with the ∼8-nm diameter of our NLRC4CARD

filament structure. Similarly, a cryo-electron tomography (cryo-ET) study of the overexpressed, NAIP-activated full-lengthNLRC4 inflammasome showed a shallow, right-handed helicalstructure with a diameter of ∼28.0 nm, 11.6 subunits per turn,and a helical pitch of 6.5 nm (31). These two structures are re-lated by a lock washer-like twist of the NLRC4NBD−LRR region.Unlike the CARD-deleted NLRC4 ring with a central hole, thefull-length shallow NLRC4 helix contains a rod-shaped volumein the center that was designated as the CARD column (31). Togain better understanding on the full-length NLRC4 assembly,we docked the NLRC4CARD filament structure and the activatedNLRC4NBD−LRR structure (PDB ID code 3JBL) (16, 17) into thecryo-ET map (Fig. 4A). Quite remarkably, at a 1:1 molecularratio, the height of the central volume for the CARD helixmatched well the height of the peripheral volume for the NBD-LRR helix (Fig. 4A). This observation is also explained by thesimilar rise per subunit for the NBD-LRR helix (∼5.7 ± 0.3 Å

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Gel Filtration Profile of NLRC4CARD Wild-type and Mutants

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Fig. 3. Structural analysis of the NLRC4CARD filament structure. (A) Surface representation of ASCCARD filament structure, side view and top view. (B)Schematic diagram of the helical filament, with three neighboring subunits highlighted in green, magenta, and cyan. (C) Detailed type I, II, and III interfaces,respectively, of the NLRC4CARD filament structure. (D) Gel filtration profile of NLRC4CARD WT and mutants. Void fractions are from elution volumes 7 mL to9 mL while less aggregated fractions are from elution volumes 14 mL to 17 mL. (E) WT and NLRC4CARD mutants overexpressed in HeLa cells examined byconfocal microscopy. (Scale bar: 10 nm.)

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per subunit) (31) and the CARD helix (5.1 Å per subunit), de-spite the different numbers of subunits per turn.It is intriguing that the helical architecture of full-length

NLRC4 provides a fairly inefficient architecture for nucleatingcaspase-1 filament formation, and the longer the helical assem-bly, the less efficient this ability. This is because each helicalassembly takes up many NLRC4 molecules, and yet only providesone nucleus to recruit and activate caspase-1. Structural analysisand experimental data show that only one end of the CARD fil-ament is preferred for caspase-1 recruitment (see below). How-ever, due to the low resolution of the CARD volume, direction ofthe CARD filament within the outer helix of NBD and LRR isambiguous. We argue that, if NLRC4 indeed forms shallow helicaloligomers at the endogenous expression level in cells, they may bevery short helices to maximize caspase-1 activation.

Structure Comparison of ASCCARD, NLRC4CARD, and Casp-1CARD Filaments.To gain deeper insights into these CARD filament assemblies, wecompared the filament structures of ASCCARD and NLRC4CARD,as well as our previously published Casp-1CARD (14). All thesefilaments share a common overall architecture with similar helicalparameters. In all cases, type III interactions mediate intrastrandassembly, and type I and II interactions mediate interstrand as-sembly (Figs. 2B and 3B). Upon closer examination of a singleturn in these CARD filaments, we found that the top and bottomsurfaces of each turn are largely charge complementary (Fig. 4B).Although the detailed features vary, they could be accounted forby the difference in the structures of the subunits. The similarity inthe charge distribution patterns among ASC, NLRC4, and caspase-1(Fig. 4 B and C) suggests a mechanism of caspase-1 recruitment bycharge complementarity.

ASCCARD and NLRC4CARD Nucleate Casp-1CARD Filament AssemblyUnidirectionally. Both ASC and NLRC4 are able to nucleate theassembly and activation of caspase-1 via homotypic CARD–

CARD interactions. To recapitulate this process, we employeda fluorescence polymerization (FP) assay to reconstitute thisprocess in vitro (Fig. 5 A–D). As controls, WT ASCCARD andNLRC4CARD efficiently promoted Casp-1CARD polymerization.As expected, mutants that disrupt ASCCARD and NLRC4CARD

filament formation also failed to promote caspase-1 polymeri-zation (Fig. 5 A–D). ASCCARD, NLRC4CARD, and Casp-1CARD

filaments share a similar helical symmetry, which indicates amolecular templating mechanism for nucleation and assembly.In other words, ASCCARD or NLRC4CARD serves as a platformto promote the assembly of Casp-1CARD along its helical tra-jectory, a mechanism also found in other death domain familycomplexes (4).We further used nano-gold labeling experiments to localize

ASC and NLRC4 in their complexes with Casp-1CARD. Weexpressed biotinylated ASCCARD and NLRC4CARD and usedthem to nucleate Casp-1CARD filaments. We then used 6-nmstreptavidin-gold to label these filaments and visualized themby negative staining EM. The experiment showed that both ASCand NLRC4 were found at only one end of Casp-1CARD fila-ments (Fig. 5 E and F), suggesting unidirectional polymerization.However, in contrast to engagement of the FADD death effectordomain (DED) unidirectionally to caspase-8 tandem DED (32),both of which are members of the death domain superfamily, allof the surfaces of ASCCARD and NLRC4CARD display chargecomplementarity with those of Casp-1CARD. Therefore, to ana-lyze in more detail why caspase-1 is recruited only to one end ofthe helical platforms of ASC or NLRC4, we calculated thepredicted buried surface areas between ASCCARD and Casp-1CARD and between NLRC4CARD and Casp-1CARD (SI Appen-dix, Table S3). The calculations showed that the buried interfacesare larger if both ASC and NLRC4 recruit Casp-1CARD fromtheir type Ib, IIb, and IIIb surfaces (Fig. 5 G and H and SI Ap-pendix, Fig. S5 and Table S3), suggesting a unified mechanismthat ASC and NLRC4 use to recruit caspase-1 through CARD–

CARD heterotypic interactions.

DiscussionHigher order assembly-mediated signal transduction has beenproposed to be a general mechanism of innate immune signaling(33). With the structure elucidation of ASCCARD and NLRC4CARD

filaments, we elaborated more details in the nucleation and po-lymerization of these higher order assemblies. First, ASCCARD

and NLRC4CARD share a similar assembly pattern, with the typeIII interface forming intrastrand interactions, and the type I and IIinterfaces forming interstrand interactions. Second, interfacesof ASCCARD and NLRC4CARD are mainly composed of charge-complementary residues. A similar assembly pattern is also true inthe case of Casp-1CARD filament (14). This observation indicatesthe possible recruitment of downstream Casp-1CARD throughcharge complementarity. Third, as upstream nucleators, bothASCCARD and NLRC4CARD must form oligomers to recruit down-stream Casp-1CARD and promote its assembly. In summary, weshowed a unified polymerization and nucleation process ofASC- and NLRC4-mediated caspse-1 assembly and activation.On the other hand, previous structural studies of hetero-

oligomeric CARD complexes showed that the upstream mole-cules always use one unique side to form a structural platform forthe recruitment of downstream molecules. We compared theproposed ASC/Casp-1 and NLRC4/Casp-1 CARD hetero-complexes with Apaf-1/Casp-9 and RIG-I/MAVS CARD het-erocomplexes. These three systems all adopt helical assembly butdisplay distinct features (Fig. 6). In the Apaf-1/Casp-9 complexcore, three Apaf-1 CARDs form one turn to recruit three Casp-9CARDs; due to the special assembly mode within the apoptosome,the complex assembly is not infinite but limited at up to a 4:4complex (34–37). All of the subunits in one turn use the type IIIinterface, and the type I and type II interactions are responsible for

A

C

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Fig. 4. Structural comparison of ASC, NLRC4, and caspase-1 CARD filaments.(A) Fitting of NLRC4CARD filament structure (light blue, PDB ID code 6DRP)and NLRC4ΔCARD structure (pink, PDB ID code 3JBL) into the NLRC4 tomog-raphy map (EMDB 2901), top view and side view. The ratio of fittedNLRC4CARD and NLRC4ΔCARD subunits is 1:1. (B) Top and bottom view com-parison of the electrostatic surface of one layer of ASC, NLRC4, and caspase-1CARD filaments, respectively. (C) Multiple sequence alignment of ASC,NLRC4, and caspase-1 CARD domains. Different colors represent differenttypes of interface (type Ia, red; type Ib, green; type IIa, purple; type IIb, cyan;type IIIa, yellow; type IIIb, blue).

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contacts between Apaf-1 and Casp-9. Different from the Apaf-1/Casp-9 complex, the RIG-I tandem CARDs (2CARD) forms alimited tetramer to mediate infinite MAVS filament assembly(38). In this tetramer, the RIG-I 2CARD subunits form type IIinteractions within subunits, and type I and III interactions

between subunits. The second CARDs in the tetramer nucleateMAVS filament formation using the type I and type II interactions.Here, we show a different assembly pattern. Both ASC and NLRC4CARDs are able to self-assemble into filaments for downstreamCasp-1CARD recruitment, with the type III interface mediating

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R160 P156

N155 T154

E152 R45

E41

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P)

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+D25K (100 nM) +E36R (100 nM) +R52E (100 nM)

+R9E (100 nM)

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filaments

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Fig. 5. Recruitment of Casp-1CARD by ASCCARD and NLRC4CARD. (A) FP assay showing Casp-1CARD filament assembly nucleated by ASCCARD WT and mutants. (B)Initial polymerization rates of Casp-1CARD nucleated by ASCCARD WT and mutants. Error bars stand for fitting error. (C) FP assay showing Casp-1CARD assemblynucleated by NLRC4CARD WT and mutants. (D) Initial polymerization rate of Casp-1CARD nucleated by NLRC4CARD WT and mutants. Error bars stand for fittingerror. (E) Gold labeling of ASC/Casp-1 CARD filament. (F) Gold labeling of NLRC4/Casp-1 CARD filament. (G) Predicted type I, type II, and type III interfaces ofASC recruitment of caspase-1. (H) Predicted type I, type II, and type III interfaces of NLRC4 recruitment of caspase-1. (Magnification: E and F, 30,000×.)

A B C

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Fig. 6. A schematic for the assembly of different CARD complexes. (A) The Apaf-1/Casp-9 CARD assembly. (B) The RIG-I/MAVS CARD assembly. (C) The ASC/Casp-1 or NLRC4/Casp-1 CARD assembly.

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interactions between neighboring subunits in one turn and type Iand type II interfaces dominating the interactions of subunitsbetween turns. The recruitment specificity between ASC andcaspase-1 and between NLRC4 and caspase-1 comes from bothcharge and shape complementarity, which is the case for theformation of many heterocomplexes in the death domain su-perfamily, including the Myddosome (39). Our study providesexamples for CARD assembly-mediated signal transduction.

Materials and MethodsProtein Expression and Purification. To generate monomeric ASCCARD, theCARD domain of human ASC (A107-S195) was cloned into an engineeredHIS-MBP-SUMO sandwich-tagged vector. This construct was transformedinto BL21(DE3) cells and expressed overnight using 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 18 °C. The cells were harvestedand lysed by sonication in lysis buffer containing 20 mM Hepes, pH 8.0,200 mM NaCl, 5 mM imidazole, 5 mM β-mercaptoethanol, and 10% glycerol.Cell lysate was then centrifuged, and the supernatant containing monomericASCCARD was incubated with nickel-nitrilotriacetic acid (Ni-NTA) affinity resinfor 1 h at 4 °C, washed in lysis buffer containing 20 mM imidazole, and elutedwith lysis buffer containing 300 mM imidazole. The eluate was subsequentlyloaded onto a Superdex 200 10/300 GL column preequilibrated with 20 mMHepes, pH 8.0, 150 mM NaCl, and 2 mM DTT. Peak fractions of monomericASCCARD were collected and treated by TEV to remove N-terminal His-MBPtag. The ASCCARD filament was formed directly after cleavage. The sample wasthen incubated with amylose resin to get rid of excess His-MBP. To assess theeffects of the interfacial residues in filament assembly, we generated the His-GFP–tagged ASCCARD, which formed filament and eluted in the void fractionsof a gel filtration column. All mutants in this construct were introduced usingthe QuikChange mutagenesis protocol and purified in a similar method.

The CARDdomain of humanNLRC4 (M1-S120)was cloned into an engineeredpET28a vector, with an N-terminal His-GFP tag. This construct was transformedinto BL21(DE3) cells and expressed overnight using 0.4 mM IPTG induction at18 °C. Similar to the purification of ASCCARD, NLRC4CARD was purified by Ni-NTAaffinity chromatography followed by gel filtration. Void fractions of NLRC4CARD

filament were collected. All mutants in this construct were introduced using theQuikChange mutagenesis protocol and purified in a similar method.

Cryo-EM Data Collection and Processing. For cryo-EM sample preparation, 3 μLof filament sample was applied to glow discharged holey carbon Quantifoilgrids (R1.2/1.3) and plunge-frozen into liquid ethane using a Vitrobot MarkIV (FEI). Movie mode micrographs were collected at the National CancerInstitute Cryo-Electron Microscopy Facility on a 300-keV FEI Titan Krioselectron microscope equipped with a K2 summit direct electron detector,under superresolution counting mode and pixel size 0.66 Å. Each movie stackcontained 40 subframes, and the exposure time for each frame was 300 ms.This resulted in an accumulated exposure time of 12 s, and dose per exposurewas ∼41 electrons per Å2. All subframes in each movie stack were subjected todrift correction and dose weighting and then added up to a single image withMotionCor2 (40). Coordinates used for filament extraction were generatedusing the program e2helixboxer within EMAN2 (41). RELION (27) was used forall of the following data-processing steps, except that a starting model wasproduced by the SPIDER (42) software package and IHRSR (26), with a sepa-rate dataset collected on a Tecnai Arctica electron microscope.

For each dataset, symmetry information was obtained by trial-and-error based on the averaged power spectrum. For the ASCCARD filament,−100.60° and 5.10 Å were found to give a stable reconstruction and lead torecognizable secondary structures. Then, 264,167 particles were firstextracted in RELION (27), with a shift of two asymmetric units for eachsegment box. After two rounds of 2D classification and one round of 3Dclassification, 226,603 particles remained for the final refinement. The re-fined helical symmetry was −100.58° and 5.00 Å. Postprocessing in RELION(27) resulted in a 3.17-Å reconstruction. For the NLRC4CARD filament,

−100.50° and 5.10 Å were found to give a stable reconstruction and lead torecognizable secondary structures. Then, 400,565 particles were firstextracted in RELION, with a shift of one asymmetric unit for each segmentbox. After two rounds of 2D classification and one round of 3D classification,199,312 particles remained for the final refinement. The refined helicalsymmetry was 100.48° and 4.93 Å. Postprocessing in RELION (27) resulted ina 3.58-Å reconstruction. In both cases, 3D classification only revealed negli-gible differences in helical symmetries, indicating mainly rigid assembly ofboth filaments (SI Appendix, Fig. S1).

Model Building and Refinement. The ASCCARD monomer structure was derivedfrom the NMR structure of ASC (PDB ID code 2KN6) (43). The NLRC4CARD

monomer structure was modeled with the SWISS-MODEL server (44) using aCasp-1CARD subunit in its filament structure (14). For each structure, a fila-ment model containing eight subunits was fitted in the EM density bymanual adjustment in Coot (29) and subsequent refinement in Phenix (45).An EM map of full-length NLRC4 was downloaded from EMDB (ID 2901).Monomeric NACHT-LRR was downloaded from PDB (PDB ID code 5AJ2) andmanually fitted in the EM map in UCSF Chimera (46). Helical symmetry wasthen imposed to generate a model with 33 NACHT-LRR molecules, and eachmolecule was fitted into the EM map separately. An NLRC4CARD filamentmodel containing 33 molecules was manually fitted in the central rod-likedensity in UCSF Chimera (46).

Fluorescence Polarization Assay. A C-terminal “LPETG” motif was added to anative N-terminal MBP-tagged ASC-CARD construct for sortase labeling (47)and a fluorescence polarization (FP) assay. For labeling, 30 μM of a freshlypurified protein substrate with the “LPETG” motif was incubated with 5 μMcalcium-independent sortase and 500 μM tetramethylrhodamine (TAMRA)-conjugated triglycine nucleophile (GGG-TAMRA) overnight at 4 °C. The mix-ture was then passed through a size-exclusion column to remove excess nu-cleophile. Labeled proteins were diluted to an appropriate concentration toperform FP assays on a SpectraMax M5e plate reader. Each experiment wasrepeated three times. Polarization values were averaged and plotted in Excel.

Cellular Imaging. ASC-mCherry and NLRC4-eGFP constructs were transfectedinto HeLa cells using standard protocols. The cells were fixed and stained byHoechst 24 h posttransfection and then examined by confocal microscopy.

Nano-Gold Labeling. ASCCARD and NLRC4CARD were cloned into the pDW363biotinylation vector to be expressed as N-terminal biotin acceptor peptide-tagged recombinant proteins, which become biotinylated by the BirA en-zyme encoded on the pDW363 vector when expressed in Escherichia coli(48). The pDW363 vector containing either ASCCARD or NLRC4CARD wascotransformed into E. coli BL21(DE3) with a pET28a vector containing His-GFP–tagged caspase-1 CARD for coexpression. The binary complexes ofASCCARD/Casp-1CARD and NLRC4CARD/Casp-1CARD were purified by Ni-NTA af-finity and gel filtration, similar to the purification of His-GFP-NLRC4CARD

described above. Streptavidin-gold conjugate (6-nm-diameter gold; ElectronMicroscopy Sciences) was employed for labeling the biotinylated binarycomplexes. A carbon-coated copper EM grid was incubated with 5 μL ofsample for 1 min at room temperature, followed by blotting with filter paperto remove excess samples. Then, the grid was rinsed for 1 min using 25 μL ofincubation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM Tris(2-carbox-yethyl)phosphine, and 0.1% gelatin) three times. The grid was incubated with25 μL of 6-nm streptavidin-gold conjugate diluted in incubation buffer for30 min at room temperature. The grid was washed three times with incubationbuffer and stained by 1% uranyl formate for examination by electron microscopy.

ACKNOWLEDGMENTS. This work was supported by US National Institutes ofHealth Grants HD087988 and AI124491 (to H.W.), by Harvard Digestive andDisease Center Grant HDDC P30 DK034854 (to T.-M.F.), and by the NationalCancer Institute’s National Cryo-EM Facility at the Frederick National Labo-ratory for Cancer Research.

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