Cryo-EM Structures of ASC and NLRC4 CARD Filaments INAUGURAL ARTICLE Reveal a Unified Mechanism of Nucleation and Activation of Caspase-1

Cryo-EM structures of ASC and NLRC4 CARD filaments INAUGURAL ARTICLE reveal a unified mechanism of nucleation and activation of caspase-1

Yang 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 Molecular Medicine, 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 complexes via its NBD and LRR and caspase-1 via its CARD upon ligand that activate caspase-1 upon sensing extrinsic microbial invasions stimulation (15–19) (Fig. 1A). As the universal effector of canon- and intrinsic sterile stress signals. During inflammasome assembly, ical inflammasomes, caspase-1 is recruited and polymerized through adaptor proteins ASC and NLRC4 recruit caspase-1 through its CARD to form filamentous structures, bringing the caspase homotypic caspase recruitment domain (CARD) interactions, lead- catalytic domains into proximity and leading to its dimerization ing to caspase-1 dimerization and activation. Activated caspase-1 and activation (14). Activated caspase-1 processes cytokines pro– processes proinflammatory cytokines and Gasdermin D to induce IL-1β and pro–IL-18 to their mature forms to elicit inflammatory cytokine maturation and pyroptotic cell death. Here, we present responses and cleaves Gasdermin D to form pores that release cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and the cytokines and cause pyroptotic cell death (20–25). ASC CARD filaments mediated by conserved three types of asym- As the first CARD filament structure in inflammasomes, metric interactions (types I, II, and III). We find that the CARDs of Casp-1CARD revealed the molecular mechanism of its self-assembly, these two adaptor proteins share a similar assembly pattern, which

as well as regulation by CARD-only proteins INCA and ICERBERG BIOCHEMISTRY matches that of the caspase-1 CARD filament whose structure we (14). However, how the upstream adaptors nucleate Casp-1CARD defined previously. These data indicate a unified mechanism for filament assembly still remains elusive. By visualizing the structures downstream caspase-1 recruitment through CARD–CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its Significance CARD and its nucleotide-binding domain (NBD), respectively. Inflammasomes are cytosolic protein complexes that detect the ASC | NLRC4 | inflammasome | caspase-1 | CARD presence of pathogens and damages to elicit immune responses, and dysregulation in inflammasome signaling is associated with s the first line of defense, the innate immune system em- many human diseases. As the unified downstream effector of – Aploys a variety of pattern recognition receptors (PRRs) to canonical inflammasomes, caspase-1 is recruited though CARD detect pathogen-associated molecular patterns (PAMPs) and CARD interactions with the adaptor proteins ASC or NLRC4. We damage-associated molecular patterns (DAMPs) (1–3). So far, at have determined the cryo-EM structures of ASC CARD and least five families of PRRs have been characterized, including NLRC4 CARD filaments. Using multidisciplinary methods, we Toll-like receptors (TLRs), RIG-I–like receptors (RLRs), C-type reveal a common mechanism of caspase-1 CARD nucleation, lectin receptors (CLRs), AIM2-like receptors (ALRs), and nucleotide- assembly, and activation by equivalent assembly patterns in ASC and NLRC4. Collectively, our data provide insights into binding domain (NBD) and leucine-rich repeat (LRR)–containing inflammasome assembly and activation and afford structural proteins (NLRs) (4). Of these, upon ligand stimulation, ALRs and platforms for modulating these CARD–CARD interactions in some NLRs have been shown to form oligomeric supramolecular potential therapeutic applications. complexes known as canonical inflammasomes, which also contain A adaptor proteins and caspase-1 (Casp-1) (2, 3) (Fig. 1 ). Exam- Author contributions: Y.L., T.-M.F., and H.W. designed research; Y.L., T.-M.F., A.L., K.W., ples of canonical inflammasomes include, but are not limited to, J.R., and C.S. performed research; Y.L., T.-M.F., A.L., and H.W. analyzed data; and Y.L., the AIM2 inflammasome, the NLRP1 inflammasome, the NLRP3 T.-M.F., and H.W. wrote the paper. inflammasome, and the NAIP inflammasomes (2). Reviewers: T.S.X., Case Western Reserve University; and R.Z., Washington University. Different inflammasomes are responsible for recognition of, The authors declare no conflict of interest. and activation by, different ligands. For example, AIM2 recog- Published under the PNAS license. nizes double-stranded DNA in the cytosol (5–7); NLRP3 re- + Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, sponds to K efflux that is in turn induced by multiple stimuli, www.wwpdb.org [PDB ID codes 6DRN (ASCCARD filaments) and 6DRP (NLRC4CARD fila- such as extracellular ATP, uric acid crystals, and the bacterial 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 toxin nigericin (8); and NAIP proteins detect flagellin and com- CARD (NLRC4 filaments)]. ponent proteins of the bacterial type III secretion system (9–12). 1Y.L. and T.-M.F. contributed equally to this study. Ligand binding activates these proteins to recruit adaptor pro- 2Present address: Department of Biophysics, University of Texas Southwestern Medical teins, such as ASC and NLRC4, which subsequently engage the Center, Dallas, TX 75390. downstream effector caspase-1. Most inflammasomes use the 3To whom correspondence may be addressed. Email: [email protected] or Tianmin. ASC adaptor, which possesses an N-terminal Pyrin domain (PYD) [email protected]. and a C-terminal caspase recruitment domain (CARD) (13) (Fig. 4Present address: Department of Biological Sciences, Ribon Therapeutics, Inc., Lexington, 1A). The N-terminal PYD interacts with the PYD of the upstream MA 02421. sensors, and the C-terminal CARD recruits caspase-1 via homo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. typic CARD–CARD interactions (13, 14). In contrast, the NLRC4 1073/pnas.1810524115/-/DCSupplemental. adaptor exists only in NAIP inflammasomes, bridging an NAIP

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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 and NLRC4 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 EM reconstruction fitted with the NLRC4CARD filament model with each subunit in a different color. One subunit is enlarged for closer view.

of ASCCARD and NLRC4CARD filaments using cryo-EM, here, structure of ASCCARD (PDB ID code 2KN6) could be easily we reveal that ASC and NLRC4 adopt the same mechanism to docked into the EM density (Fig. 1D and SI Appendix, Table S1). nucleate the assembly and activation of caspase-1. The ASCCARD The model was manually adjusted in Coot (29), followed by and NLRC4CARD filament structures show assembly patterns refinement in Phenix (30). The amino acid sequence was un- similartothatofCasp-1CARD, indicating that these adaptors ambiguously registered due to the clearly defined side chains. template the polymerization of Casp-1CARD. Further structural There is almost no density outside the filament, indicating that the analyses and biochemical assays show that ASC and NLRC4 C-terminal SUMO tag was largely disordered (Fig. 1D). The utilize similar interfaces to recruit caspase-1 and confer a uni- resolution of this reconstruction was measured at 3.2 Å using the directional polymerization of Casp-1CARD by charge and shape gold standard Fourier shell correlation (FSC) in RELION (SI complementarity. Appendix,Fig.S3). In the case of the NLRC4CARD domain, we found that the Results construct of GFP-NLRC4CARD directly forms filaments suitable Cryo-EM Reconstruction of the ASCCARD and NLRC4CARD Filament for structure determination (Fig. 1 B and E). As the cryo-EM Structures. Both ASCCARD and NLRC4CARD are capable of nu- images showed, NLRC4CARD filaments are generally shorter and cleating the assembly and activation of caspase-1 (Fig. 1A). To wider than ASCCARD filaments, likely due to the effect of the gain a mechanistic understanding of this process, we prepared larger GFP tag (Fig. 1 C and E). We employed a similar strategy ASCCARD and NLRC4CARD filaments for cryo-EM study. We as used for ASCCARD to determine the NLRC4CARD filament found that His-MBP-ASCCARD-SUMO was purified as monomers structure. The helical symmetry was first calculated from the over a gel filtration column and ASCCARD-SUMO formed filaments averaged power spectrum (SI Appendix, Fig. S2B)as−100.50° in upon proteolytic removal of the His-MBP tag by the Tobacco Etch azimuthal angle and 5.10 Å in axial rise per subunit, which was Virus (TEV) protease (Fig. 1 B and C). The averaged power refined to −100.48° and 4.93 Å, respectively. The GFP tag is spectrum showed a similar diffraction pattern to that of Casp-1CARD largely disordered in that the final reconstruction contains only filaments (14). Based on possible indexing of the power spectrum, weak noisy densities in the periphery. We also calculated the we adopted a calculated one-start helical symmetry with an azi- power spectrum from the final volume, which matched well with muthal angle of −100.60° and an axial rise of 5.10 Å per subunit. the experimental power spectrum (SI Appendix, Fig. S2B). A The iterative helical real-space reconstruction (IHRSR) method homology structure model of NLRC4CARD derived from the (26) was used to generate an intermediate map, starting from a Casp-1CARD structure (PDB ID code 5FNA) (14) was readily fitted solid cylinder as the initial model. This map was then used as into the cryo-EM density (SI Appendix,TableS2and Fig. 1F). an initial model in RELION (27) for 3D classification (SI Ap- Similar to the case for ASCCARD, the obvious side chain densities pendix,Fig.S1) and refinement. The final volume contains of the NLRC4CARD map enabled manual model building in Coot mostly α-helices with the refined helical symmetry parameters of (29), followed by refinement in Phenix (30). The resolution was −100.58° rotation and 5.00 Å translation per subunit, respectively. measured at 3.6 Å using gold standard FSC (SI Appendix,Fig.S4). Each subunit showed the typical six α-helices arranged in a W shape, which is a common feature of the death domain super- Structure of the ASCCARD Filament. The diameter of the ASCCARD family that includes the CARD (28) (Fig. 1D). The calculated filament is ∼8 nm, with a central hole of less than 1 nm (Fig. 2A). power spectrum from the final volume corresponded well with the The filament structure assembles through a left-handed one-start experimental power spectrum (SI Appendix,Fig.S2A). The NMR helical symmetry, with about 3.6 subunits per turn (Fig. 2B). Like

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1810524115 Li et al. Downloaded by guest on September 23, 2021 other filaments formed by members of the death domain su- To validate the importance of the interfacial residues identi- CARD perfamily, formation of the ASC filament is mediated by fied by our structural analysis, we generated site-directed mutants INAUGURAL ARTICLE three types of asymmetric interactions, namely type I, type II, on a construct of ASCCARD fused to GFP (GFP-ASCCARD). WT and type III interactions (28) (Fig. 2B). In the filament archi- GFP-tagged ASCCARD primarily eluted at the void fraction on a tecture, the type III interaction is along the direction of the one- gel filtration column (Fig. 2D). In contrast, R119D, N128A/E130R, start helical strand while type I and type II interactions generate and D134K of type I mutations completely abolished filament connections between the adjacent turns of the helical strands formation (Fig. 2D). The effectiveness of these charge-reversal (Fig. 2B). The type I interaction is mostly composed of charge– mutations confirmed our observation that type I interaction is charge interactions between residues located on helix α2 of one dominated by charge–charge interactions. In the case of type II molecule and helices α1 and α4 of the partner molecule and is interactions, W169G, Y187A, Y187K mutations almost com- the most extensive in surface area among the three types of in- pletely disrupted filament formation (Fig. 2D). Additionally, teractions. Possible residues involved in this interaction include mutation of Y187 to L or H partially disrupted filament for- R119, E130, D134, and R160 (Fig. 2C). These residues form mation, further showing the hydrophobic interaction of the type several electrostatically complementary pairs. Unlike the type I II interface (Fig. 2D). D143K/E144K and R160E of type III in- interaction, the type II interaction in the ASCCARD filament is teractions almost completely disrupted filament formation (Fig. mainly contributed by hydrophobic residues, including W169 and 2D). We further examined mCherry-tagged ASCCARD mutants Y187 (Fig. 2C). The type III interaction is also dominated by using confocal microscopy in HeLa cells. In line with our in vitro charge–charge interactions, with R160 of helix α4 and D143 and biochemical data, WT ASCCARD formed filaments in cells while E144 of helix α3 forming charge complementary pairs at the mutations that proved to be disruptive in vitro also abolished fil- interface (Fig. 2C). ament formation in cells (Fig. 2E). These mutagenesis studies

A C ASCCARD filament Ia IIa IIIa

V140 E144 R160 Q145 BIOCHEMISTRY R119 W169 D143 E130 S164 P167 N170 T142 R160 D134 T127 Q147 Y137 Y187 N128 P156 10 Å D143 R125 T154 R150 Y146 D191 Q185 N155 80 Å Ib IIb IIIb B D E Gel Filtration Profile of ASCCARD Wild-type and Mutants Hoechst mCherry BF Merge Void Less aggregated ASCCARD WT fractions fractions 10 nm Wild-type (WT)

IIIa IIIb R119D (Type Ia) R125E N128A/E130R (Type Ib)

D134K (Type Ib) D134K Interactions in the ASCCARD filament R125E (Type IIb) Type II Type I W169G (Type IIa) R119D

Y187A (Type IIb) N128A/ Y187K (Type IIb) E130R

Y187L (Type IIb) W169G Y187H (Type IIb) Helical axis D143K/E144K Type III (Type IIIa) helical-strand D143K/ direction R160E (Type IIIb) E144K

Fig. 2. Structural analysis of the ASCCARD 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 ASCCARD filament structure. (D) Gel filtration profile of ASCCARD WT and mutants. Void fractions are from elution volumes 7 mL to 9 mL while less aggregated fractions are from elution volumes 14 mL to 17 mL. (E) WT and ASCCARD mutants overexpressed in HeLa cells examined by confocal microscopy. (Scale bar: 10 nm.)

Li et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 23, 2021 strongly support the correctness of our structural model and observation in vitro, WT NLRC4CARD formed filaments in cells, analysis. and surface mutants of all three types of interactions abolished or attenuated filament formation (Fig. 3E). Collectively, these mu- CARD Structure of the NLRC4CARD Filament. The NLRC4 filament tagenesis data further support the helical assembly model of the has a very similar architecture to the ASCCARD filament, with a NLRC4CARD filament. diameter of ∼8 nm and an even smaller central hole (Figs. 2A and 3A). Like the ASCCARD filament, each subunit of the The NLRC4CARD Filament Exists in the Full-Length NLRC4 Structure. NLRC4CARD filament interacts with its neighboring molecules Upon activation by a ligand-bound NAIP protein (18, 19), through three types of interactions, of which the type III in- NLRC4 with the N-terminal CARD deleted forms mainly 11- to teraction mediates the intrastrand contact and type I and type II 12-folded disk-shaped complexes with a central hole of ∼8nmin interactions mediate interstrand contacts (Fig. 3 A and B). The diameter and an outer diameter of ∼30 nm (16, 17). The size of type I interaction is composed of helix α2 of one molecule and the hole is compatible with the ∼8-nm diameter of our NLRC4CARD helices α1 and α4 of the other. Electrostatic complementary filament structure. Similarly, a cryo-electron tomography (cryo- residues of R9, D25, D26, and R52 form charge–charge inter- ET) study of the overexpressed, NAIP-activated full-length actions at the interface (Fig. 3C). At the type II interface, K60/ NLRC4 inflammasome showed a shallow, right-handed helical K61 and E47 interact with each other through charge comple- structure with a diameter of ∼28.0 nm, 11.6 subunits per turn, mentarity (Fig. 3C). Like the type I interface, the type III in- and a helical pitch of 6.5 nm (31). These two structures are re- − terface is mainly composed of hydrophilic residues, including the lated by a lock washer-like twist of the NLRC4NBD LRR region. charge pair consisting of K45 and E44 (Fig. 3C). Unlike the CARD-deleted NLRC4 ring with a central hole, the To further validate the NLRC4CARD filament model, we full-length shallow NLRC4 helix contains a rod-shaped volume performed site-directed mutagenesis on the GFP tagged con- in the center that was designated as the CARD column (31). To struct. While WT GFP-NLRC4CARD mainly eluted at the void gain better understanding on the full-length NLRC4 assembly, fraction, mutations of residues on type I and type III interfaces we docked the NLRC4CARD filament structure and the activated − (R52E on type Ia, D25K on type Ib, and E36R on type IIIa) NLRC4NBD LRR structure (PDB ID code 3JBL) (16, 17) into the effectively abolished its aggregation ability (Fig. 3D). R9E on cryo-ET map (Fig. 4A). Quite remarkably, at a 1:1 molecular type Ia and K60E/K61D double mutation on the type IIa in- ratio, the height of the central volume for the CARD helix terface led to partial disruption of filament formation (Fig. 3D). matched well the height of the peripheral volume for the NBD- We also investigated eGFP-tagged NLRC4CARD mutants us- LRR helix (Fig. 4A). This observation is also explained by the ing confocal microscopy in HeLa cells. Consistent with our similar rise per subunit for the NBD-LRR helix (∼5.7 ± 0.3 Å

A C NLRC4CARD Filament Ia IIa IIIa E44

D49 Q48 E36 C43 Q13 E37 N34 K45 H56 K61 Q22 N39 R52 S8 T18 V46 R9 S63 K60 E47 K21 E47 Q22 D25 K5 N77 W76 E36 R35 Q48 D26 F28 D83 Q13 80 Å Ib IIb IIIb

B Interactions in the D E CARD Hoechst eGFP BF Merge NLRC4 filament Gel Filtration Profile of NLRC4CARD Wild-type and Mutants Less aggregated WT NLRC4CARD Void fractions 10 nm fractions

Wild-type (WT) R9E IIIa IIIb R9E (Type Ia) R52E Type II Type I R52E (Type Ia) D25K D25K (Type Ib) Type III K60E/ helical- K60E/K61D (Type IIa) K61D Helical strand axis direction E36R (Type IIIa) E36R

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 to 9 mL while less aggregated fractions are from elution volumes 14 mL to 17 mL. (E) WT and NLRC4CARD mutants overexpressed in HeLa cells examined by confocal microscopy. (Scale bar: 10 nm.)

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1810524115 Li et al. Downloaded by guest on September 23, 2021 A B CARD interactions. To recapitulate this process, we employed

a fluorescence polymerization (FP) assay to reconstitute this INAUGURAL ARTICLE process in vitro (Fig. 5 A–D). As controls, WT ASCCARD and NLRC4CARD efficiently promoted Casp-1CARD polymerization. As expected, mutants that disrupt ASCCARD and NLRC4CARD filament formation also failed to promote caspase-1 polymerization (Fig. 5 A–D). ASCCARD, NLRC4CARD, and Casp-1CARD filaments share a similar helical symmetry, which indicates a molecular templating mechanism for nucleation and assembly. In other words, ASCCARD or NLRC4CARD serves as a platform to promote the assembly of Casp-1CARD along its helical tra- jectory, a mechanism also found in other death domain family complexes (4). We further used nano-gold labeling experiments to localize C ASC and NLRC4 in their complexes with Casp-1CARD.We expressed biotinylated ASCCARD and NLRC4CARD and used them to nucleate Casp-1CARD filaments. We then used 6-nm streptavidin-gold to label these filaments and visualized them by negative staining EM. The experiment showed that both ASC and NLRC4 were found at only one end of Casp-1CARD filaments (Fig. 5 E and F), suggesting unidirectional polymerization. However, in contrast to engagement of the FADD death effector Fig. 4. Structural comparison of ASC, NLRC4, and caspase-1 CARD filaments. domain (DED) unidirectionally to caspase-8 tandem DED (32), (A) Fitting of NLRC4CARD filament structure (light blue, PDB ID code 6DRP) both of which are members of the death domain superfamily, all Δ CARD CARD and NLRC4 CARD structure (pink, PDB ID code 3JBL) into the NLRC4 tomog- of the surfaces of ASC and NLRC4 display charge raphy map (EMDB 2901), top view and side view. The ratio of fitted complementarity with those of Casp-1CARD. Therefore, to ana- Δ NLRC4CARD and NLRC4 CARD subunits is 1:1. (B) Top and bottom view com- lyze in more detail why caspase-1 is recruited only to one end of

parison of the electrostatic surface of one layer of ASC, NLRC4, and caspase-1 the helical platforms of ASC or NLRC4, we calculated the BIOCHEMISTRY CARD filaments, respectively. (C) Multiple sequence alignment of ASC, predicted buried surface areas between ASCCARD and Casp- NLRC4, and caspase-1 CARD domains. Different colors represent different 1CARD and between NLRC4CARD and Casp-1CARD (SI Appen- types of interface (type Ia, red; type Ib, green; type IIa, purple; type IIb, cyan; dix type IIIa, yellow; type IIIb, blue). , Table S3). The calculations showed that the buried interfaces are larger if both ASC and NLRC4 recruit Casp-1CARD from their type Ib, IIb, and IIIb surfaces (Fig. 5 G and H and SI Ap- per subunit) (31) and the CARD helix (5.1 Å per subunit), de- pendix, Fig. S5 and Table S3), suggesting a unified mechanism spite the different numbers of subunits per turn. that ASC and NLRC4 use to recruit caspase-1 through CARD– It is intriguing that the helical architecture of full-length CARD heterotypic interactions. NLRC4 provides a fairly inefficient architecture for nucleating caspase-1 filament formation, and the longer the helical assem- Discussion bly, the less efficient this ability. This is because each helical Higher order assembly-mediated signal transduction has been assembly takes up many NLRC4 molecules, and yet only provides proposed to be a general mechanism of innate immune signaling CARD CARD one nucleus to recruit and activate caspase-1. Structural analysis (33). With the structure elucidation of ASC and NLRC4 and experimental data show that only one end of the CARD fil- filaments, we elaborated more details in the nucleation and po- CARD ament is preferred for caspase-1 recruitment (see below). How- lymerization of these higher order assemblies. First, ASC CARD ever, due to the low resolution of the CARD volume, direction of and NLRC4 share a similar assembly pattern, with the type the CARD filament within the outer helix of NBD and LRR is III interface forming intrastrand interactions, and the type I and II ambiguous. We argue that, if NLRC4 indeed forms shallow helical interfaces forming interstrand interactions. Second, interfaces CARD CARD oligomers at the endogenous expression level in cells, they may be of ASC and NLRC4 are mainly composed of charge- very short helices to maximize caspase-1 activation. complementary residues. A similar assembly pattern is also true in the case of Casp-1CARD filament (14). This observation indicates CARD Structure Comparison of ASCCARD,NLRC4CARD,andCasp-1CARD Filaments. the possible recruitment of downstream Casp-1 through To gain deeper insights into these CARD filament assemblies, we charge complementarity. Third, as upstream nucleators, both compared the filament structures of ASCCARD and NLRC4CARD, ASCCARD and NLRC4CARD must form oligomers to recruit down- as well as our previously published Casp-1CARD (14). All these stream Casp-1CARD and promote its assembly. In summary, we filaments share a common overall architecture with similar helical showed a unified polymerization and nucleation process of parameters. In all cases, type III interactions mediate intrastrand ASC- and NLRC4-mediated caspse-1 assembly and activation. assembly, and type I and II interactions mediate interstrand as- On the other hand, previous structural studies of hetero- sembly (Figs. 2B and 3B). Upon closer examination of a single oligomeric CARD complexes showed that the upstream mole- turn in these CARD filaments, we found that the top and bottom cules always use one unique side to form a structural platform for surfaces of each turn are largely charge complementary (Fig. 4B). the recruitment of downstream molecules. We compared the Although the detailed features vary, they could be accounted for proposed ASC/Casp-1 and NLRC4/Casp-1 CARD hetero- by the difference in the structures of the subunits. The similarity in complexes with Apaf-1/Casp-9 and RIG-I/MAVS CARD het- the charge distribution patterns among ASC, NLRC4, and caspase-1 erocomplexes. These three systems all adopt helical assembly but (Fig. 4 B and C) suggests a mechanism of caspase-1 recruitment by display distinct features (Fig. 6). In the Apaf-1/Casp-9 complex charge complementarity. core, three Apaf-1 CARDs form one turn to recruit three Casp-9 CARDs; due to the special assembly mode within the apoptosome, ASCCARD and NLRC4CARD Nucleate Casp-1CARD Filament Assembly the complex assembly is not infinite but limited at up to a 4:4 Unidirectionally. Both ASC and NLRC4 are able to nucleate the complex (34–37). All of the subunits in one turn use the type III assembly and activation of caspase-1 via homotypic CARD– interface, and the type I and type II interactions are responsible for

Li et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 23, 2021 A B 5 E 180 CARD 160 Casp-1 (1.6 µM) 4 +WT ASCCARD (100 nM) 140 + R125E (100 nM) 3 +D134K (100 nM) 120 + W169G (100 nM) +Y187A (100 nM) 2 100 + R160E (100 nM) FP/Time (mP/min) FP/Time FP (mP) FP 1 80

60 Initial 0 Streptavidin-gold labeling of 40 Biotin-ASCCARD/Casp-1CARD 0 10 20 30 40 50 60 filaments Time (min) C D 8 F 250 7 6 200 5 Casp-1CARD (1.6 µM) CARD 4 150 +WT NLRC4 (100 nM) +R14E (100 nM) 3 +K60E/K61D (100 nM) 100 (mP/min) FP/Time 2

FP (mP) FP +R9E (100 nM) +D25K (100 nM) 1 +E36R (100 nM) 50 0

+R52E (100 nM) Initial Streptavidin-gold labeling of Biotin-NLRC4CARD/Casp-1CARD 0 10 20 30 40 50 60 filaments G Time (min) H IIb IIa Ib Ia IIb IIa Ib D143 Ia IIIb IIIa IIIb IIIa D191 Q67 F28 P79 K7 K7 R45 Y187 K64 E144 K37 E47 R160 D134 R125 K64 E38 N39 K11 Q147 R55 Q185 P156 N77 E41 D25 M15 Q48 T127 P63 N155 T154 R55 N36 K11 E41 N128 Q22 M17 W131 N36 E38 E152 R45 R15 R15 K21 K37 Q13 W76 D52 E38 ASC Casp-1 ASC Casp-1 ASC Casp-1 NLRC4 Casp-1 NLRC4 Casp-1 NLRC4 Casp-1 Predicted preferred interfaces between ASC and Caspase-1 Predicted preferred interfaces between NLRC4 and Caspase-1

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 assembly nucleated by NLRC4CARD WT and mutants. (D) Initial polymerization rate of Casp-1CARD nucleated by NLRC4CARD WT and mutants. Error bars stand for fitting error. (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 of ASC 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×.)

contacts between Apaf-1 and Casp-9. Different from the Apaf-1/ between subunits. The second CARDs in the tetramer nucleate Casp-9 complex, the RIG-I tandem CARDs (2CARD) forms a MAVSfilamentformationusingthetypeIandtypeIIinteractions. limited tetramer to mediate infinite MAVS filament assembly Here, we show a different assembly pattern. Both ASC and NLRC4 (38). In this tetramer, the RIG-I 2CARD subunits form type II CARDs are able to self-assemble into filaments for downstream interactions within subunits, and type I and III interactions Casp-1CARD recruitment, with the type III interface mediating

A B C

Casp-9 MAVS Apaf-1 IIa Casp-1 IIIa IIb RIG-I IIIb st 2 IIa ASC or CARD IIb NLRC4 IIa IIIa IIb RIG-I IIIb IIIa st IIIb 1 CARD

ASCCARD/Casp-1CARD or CARD CARD 2CARD CARD Apaf-1 /Casp-9 RIG-I /MAVS NLRC4CARD/Casp-1CARD

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.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1810524115 Li et al. Downloaded by guest on September 23, 2021 interactions between neighboring subunits in one turn and type I −100.50° and 5.10 Å were found to give a stable reconstruction and lead to recognizable secondary structures. Then, 400,565 particles were first and type II interfaces dominating the interactions of subunits INAUGURAL ARTICLE between turns. The recruitment specificity between ASC and extracted in RELION, with a shift of one asymmetric unit for each segment caspase-1 and between NLRC4 and caspase-1 comes from both box. After two rounds of 2D classification and one round of 3D classification, 199,312 particles remained for the final refinement. The refined helical charge and shape complementarity, which is the case for the symmetry was 100.48° and 4.93 Å. Postprocessing in RELION (27) resulted in formation of many heterocomplexes in the death domain su- a 3.58-Å reconstruction. In both cases, 3D classification only revealed negli- perfamily, including the Myddosome (39). Our study provides gible differences in helical symmetries, indicating mainly rigid assembly of examples for CARD assembly-mediated signal transduction. both filaments (SI Appendix, Fig. S1).

Materials and Methods Model Building and Refinement. The ASCCARD monomer structure was derived Protein Expression and Purification. To generate monomeric ASCCARD,the from the NMR structure of ASC (PDB ID code 2KN6) (43). The NLRC4CARD CARD domain of human ASC (A107-S195) was cloned into an engineered monomer structure was modeled with the SWISS-MODEL server (44) using a HIS-MBP-SUMO sandwich-tagged vector. This construct was transformed Casp-1CARD subunit in its filament structure (14). For each structure, a fila- into BL21(DE3) cells and expressed overnight using 0.4 mM Isopropyl β-D-1- ment model containing eight subunits was fitted in the EM density by thiogalactopyranoside (IPTG) induction at 18 °C. The cells were harvested manual adjustment in Coot (29) and subsequent refinement in Phenix (45). and lysed by sonication in lysis buffer containing 20 mM Hepes, pH 8.0, An EM map of full-length NLRC4 was downloaded from EMDB (ID 2901). 200 mM NaCl, 5 mM imidazole, 5 mM β-mercaptoethanol, and 10% glycerol. Monomeric NACHT-LRR was downloaded from PDB (PDB ID code 5AJ2) and Cell lysate was then centrifuged, and the supernatant containing monomeric manually fitted in the EM map in UCSF Chimera (46). Helical symmetry was ASCCARD was incubated with nickel-nitrilotriacetic acid (Ni-NTA) affinity resin then imposed to generate a model with 33 NACHT-LRR molecules, and each for 1 h at 4 °C, washed in lysis buffer containing 20 mM imidazole, and eluted molecule was fitted into the EM map separately. An NLRC4CARD filament with lysis buffer containing 300 mM imidazole. The eluate was subsequently model containing 33 molecules was manually fitted in the central rod-like loaded onto a Superdex 200 10/300 GL column preequilibrated with 20 mM density in UCSF Chimera (46). Hepes, pH 8.0, 150 mM NaCl, and 2 mM DTT. Peak fractions of monomeric CARD ASC were collected and treated by TEV to remove N-terminal His-MBP Fluorescence Polarization Assay. A C-terminal “LPETG” motif was added to a CARD tag. The ASC filament was formed directly after cleavage. The sample was native N-terminal MBP-tagged ASC-CARD construct for sortase labeling (47) then incubated with amylose resin to get rid of excess His-MBP. To assess the and a fluorescence polarization (FP) assay. For labeling, 30 μM of a freshly effects of the interfacial residues in filament assembly, we generated the His- purified protein substrate with the “LPETG” motif was incubated with 5 μM CARD GFP–tagged ASC , which formed filament and eluted in the void fractions calcium-independent sortase and 500 μM tetramethylrhodamine (TAMRA)- of a gel filtration column. All mutants in this construct were introduced using conjugated triglycine nucleophile (GGG-TAMRA) overnight at 4 °C. The mix-

the QuikChange mutagenesis protocol and purified in a similar method. ture was then passed through a size-exclusion column to remove excess nu- BIOCHEMISTRY The CARD domain of human NLRC4 (M1-S120) was cloned into an engineered cleophile. Labeled proteins were diluted to an appropriate concentration to pET28a vector, with an N-terminal His-GFP tag. This construct was transformed perform FP assays on a SpectraMax M5e plate reader. Each experiment was into BL21(DE3) cells and expressed overnight using 0.4 mM IPTG induction at repeated three times. Polarization values were averaged and plotted in Excel. 18 °C. Similar to the purification of ASCCARD, NLRC4CARD was purified by Ni-NTA CARD affinity chromatography followed by gel filtration. Void fractions of NLRC4 Cellular Imaging. ASC-mCherry and NLRC4-eGFP constructs were transfected filament were collected. All mutants in this construct were introduced using the into HeLa cells using standard protocols. The cells were fixed and stained by QuikChange mutagenesis protocol and purified in a similar method. Hoechst 24 h posttransfection and then examined by confocal microscopy.

μ Cryo-EM Data Collection and Processing. For cryo-EM sample preparation, 3 L Nano-Gold Labeling. ASCCARD and NLRC4CARD were cloned into the pDW363 of filament sample was applied to glow discharged holey carbon Quantifoil biotinylation vector to be expressed as N-terminal biotin acceptor peptide- grids (R1.2/1.3) and plunge-frozen into liquid ethane using a Vitrobot Mark tagged recombinant proteins, which become biotinylated by the BirA en- IV (FEI). Movie mode micrographs were collected at the National Cancer zyme encoded on the pDW363 vector when expressed in Escherichia coli Institute Cryo-Electron Microscopy Facility on a 300-keV FEI Titan Krios (48). The pDW363 vector containing either ASCCARD or NLRC4CARD was electron microscope equipped with a K2 summit direct electron detector, cotransformed into E. coli BL21(DE3) with a pET28a vector containing His- under superresolution counting mode and pixel size 0.66 Å. Each movie stack GFP–tagged caspase-1 CARD for coexpression. The binary complexes of contained 40 subframes, and the exposure time for each frame was 300 ms. ASCCARD/Casp-1CARD and NLRC4CARD/Casp-1CARD were purified by Ni-NTA af- This resulted in an accumulated exposure time of 12 s, and dose per exposure finity and gel filtration, similar to the purification of His-GFP-NLRC4CARD ∼ 2 was 41 electrons per Å . All subframes in each movie stack were subjected to described above. Streptavidin-gold conjugate (6-nm-diameter gold; Electron drift correction and dose weighting and then added up to a single image with Microscopy Sciences) was employed for labeling the biotinylated binary MotionCor2 (40). Coordinates used for filament extraction were generated complexes. A carbon-coated copper EM grid was incubated with 5 μLof using the program e2helixboxer within EMAN2 (41). RELION (27) was used for sample for 1 min at room temperature, followed by blotting with filter paper all of the following data-processing steps, except that a starting model was to remove excess samples. Then, the grid was rinsed for 1 min using 25 μLof produced by the SPIDER (42) software package and IHRSR (26), with a sepa- incubation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM Tris(2-carbox- rate dataset collected on a Tecnai Arctica electron microscope. yethyl)phosphine, and 0.1% gelatin) three times. The grid was incubated with For each dataset, symmetry information was obtained by trial-and- 25 μL of 6-nm streptavidin-gold conjugate diluted in incubation buffer for CARD error based on the averaged power spectrum. For the ASC filament, 30 min at room temperature. The grid was washed three times with incubation − 100.60° and 5.10 Å were found to give a stable reconstruction and lead to buffer and stained by 1% uranyl formate for examination by electron microscopy. recognizable secondary structures. Then, 264,167 particles were first extracted in RELION (27), with a shift of two asymmetric units for each ACKNOWLEDGMENTS. This work was supported by US National Institutes of segment box. After two rounds of 2D classification and one round of 3D Health Grants HD087988 and AI124491 (to H.W.), by Harvard Digestive and classification, 226,603 particles remained for the final refinement. The re- Disease Center Grant HDDC P30 DK034854 (to T.-M.F.), and by the National fined helical symmetry was −100.58° and 5.00 Å. Postprocessing in RELION Cancer Institute’s National Cryo-EM Facility at the Frederick National Labo- (27) resulted in a 3.17-Å reconstruction. For the NLRC4CARD filament, ratory for Cancer Research.

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