Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9

The caspase recruitment domain–containing protein 9 (CARD9)–B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)–CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity—a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9–CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9–CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9–CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9–Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.

related cardiac hypertrophy (ORCH) in mice maintained on a high-fat diet. They further found that Zn 2ϩ modulates ORCH development in this context, with Zn 2ϩ deficiency aggravating and Zn 2ϩ supplementation mitigating disease severity in a Bcl10-dependent manner (18). These findings fit within a larger body of literature indicating that Zn 2ϩ acts as a signaling molecule in a number of immune cell types, wherein transient increases in cytosolic Zn 2ϩ , known as Zn 2ϩ -waves, serve to activate downstream signaling pathways (19 -22). In general, however, the targets of the Zn 2ϩ -wave and mechanisms by which Zn 2ϩ impacts signaling remain poorly understood. Similarly, despite the broad importance of CARD9, CARD9 itself has remained structurally uncharacterized, and a mechanistic understanding of its regulation is lacking.
CARD9 comprises an N-terminal CARD followed by a "coiled-coil" domain of ϳ450 amino acids containing multiple distinct regions predicted to have high coiled-coil propensity. The CARD is critical for CARD9's recruitment of Bcl10, and by homology to CARD9's closest paralogue, CARD11 (CARD11, caspase recruitment domain-containing protein 11; also known as CARMA1), the CARD9 -CARD is thought to act in a templating manner by forming a helical assembly able to potentiate the subsequent polymerization of Bcl10 required for signal propagation (6,23). We therefore hypothesize that CARD9 signaling may be regulated by modulating the accessibility of its CARD and/or its propensity to generate a helical template. To better understand molecular mechanisms underlying CARD9 function, we determined the NMR solution structure of the CARD9 -CARD, and we found, surprisingly, that it binds to Zn 2ϩ , exhibiting a dissociation constant comparable with estimates of the "free" cytosolic Zn 2ϩ concentration. Although the ground-state structure of the CARD9 -CARD is essentially identical in the apo and Zn 2ϩ -bound states, Zn 2ϩ binding strongly stabilizes the fold and reduces conformational "breathing" of the helices. Upon overexpression in Escherichia coli, the CARD9 -CARD is also capable of forming an extensively domain-swapped dimer, with interconversion of the CARD monomer and dimer strongly inhibited by Zn 2ϩ binding. Furthermore, Zn 2ϩ binding inhibits formation of helical filaments by the CARD9 -CARD monomer that otherwise spontaneously assembles in vitro. A 20-Å negative-stain EM (NS-EM) structure of these filaments reported here demonstrates that they adopt a similar symmetry as the Bcl10 -CARD helical assembly. Finally, we show through both a bulk assay and direct NS-EM visualization that the CARD9 -CARD helical assembly is capable of directly templating Bcl10 polymerization.

Results
The CARD9 -CARD binds Zn 2؉ We purified 15 N-labeled CARD9 -CARD (residues 2-97) using a cleavable affinity tag and size-exclusion chromatography (see under "Experimental procedures"). A 15 N HSQC NMR spectrum of the resulting monomeric CARD exhibits significant peak dispersion, suggesting that the domain adopts a wellfolded structure. We determined near-complete backbone and side-chain assignments for the CARD and calculated a solution structure with a backbone RMSD of 0.7 Å (RMSD calculated for structured residues 10 -97, see Table 1 for complete statistics). The CARD9 -CARD adopts a canonical death-domain structure containing six antiparallel ␣-helices with 9 N-terminal residues remaining largely unstructured. As shown in Fig. 1A, the CARD9 -CARD aligns well with the crystal structure of its closest paralogue, the human CARD11-CARD (1.9-Å backbone RMSD to PDB code 4LWD (23)), including the conserved kink in the ␣1 helix common among CARDs. The largest differences between the CARD9 and CARD11-CARDs are apparent in ␣helices ␣3 and ␣4 and the flexible ␣3-␣4 loop, a region with high B-factors in the CARD11-CARD structure and also comprising the largest sequence divergence between the CARDs.
Given the role of Zn 2ϩ in modulating ORCH and the central role of CARD9 in this pathology (18), we tested whether the CARD9 -CARD itself interacts with Zn 2ϩ . The 15 N HSQC NMR spectrum of the CARD is significantly perturbed upon addition of 1:0.5 or 1:1 concentrations of ZnCl 2 , with all shifted peaks in the slow-exchange limit, suggesting that the CARD binds Zn 2ϩ with sub-micromolar affinity (Fig. 1B). Further increasing the Zn 2ϩ concentration above a 1:1 ratio minimally affects the spectrum, indicating that the CARD9 -CARD con- tains a single high-affinity metal-binding site (Fig. S1). To determine whether this binding is specific for Zn 2ϩ , we incubated the CARD9 -CARD with other divalent metal ions. No chemical shift changes are observed upon addition of 1:10 molar concentrations of either CaCl 2 or MgCl 2 , indicating that the CARD is unable to bind Ca 2ϩ or Mg 2ϩ (Fig. 1C). A stoichiometric concentration of NiCl 2 likewise induces no chemical shift changes in the CARD9 -CARD. However, Mn 2ϩ and Cu 2ϩ do interact with the CARD9 -CARD at stoichiometric concentrations, with Cu 2ϩ inducing chemical shift changes and the paramagnetic Mn 2ϩ inducing enhanced relaxation of a number of peaks (Fig. 1C). Upon simultaneous addition of Zn 2ϩ and Mn 2ϩ to the CARD9 -CARD in a 1:1:1 ratio, the peak signature indicates that all of the CARD is Zn 2ϩ -bound, with remaining peak disappearances attributable to secondary Mn 2ϩ interactions outside the Zn 2ϩ -binding site (Fig. 1C, see arrows indicating peaks unique to the Zn 2ϩ -bound CARD9 -CARD). Simultaneous addition of stoichiometric concentrations of Cu 2ϩ and Zn 2ϩ results in populations of the CARD9 -CARD bound to each of the two metals, shown by the presence of peaks corresponding to both the Cu 2ϩ -bound and Zn 2ϩ -bound states. The Zn 2ϩ -bound peaks are ϳ50% less intense than when Zn 2ϩ is added alone (Fig. 1C, arrowheads), indicating approximately equal affinity of the CARD for Zn 2ϩ and Cu 2ϩ . These findings are consistent with the relative affinities expected by the Irving-Williams series (24). The concentration of labile cytosolic copper has proven difficult to measure with high accuracy, although estimates suggest that it is in the femtomolar range or lower (25,26). Because the CARD9 -CARD dissociation constant for Zn 2ϩ (and therefore for Cu 2ϩ ) is several orders of magnitude larger than the typical cytosolic Cu 2ϩ concentration, but comparable with estimates of the "free" cytosolic Zn 2ϩ concentration (see below), we suggest that Zn 2ϩ is likely to be a physiological ligand for CARD9, but depending on the cellular context, Cu 2ϩ binding could play a role as well. We thus proceeded to characterize Zn 2ϩ binding to the CARD9 -CARD.
Cysteine and histidine residues typically mediate Zn 2ϩ coordination, with additional binding often provided by glutamate and aspartate side chains. To determine which residues in the CARD9 -CARD are responsible for Zn 2ϩ coordination, we generated alanine substitutions at either of the two cysteines (CARD C10A or CARD C37A ) or the sole histidine (CARD H73A ),  15 N HSQC spectra of the CARD9 -CARD in the absence (black) and presence (red) of equimolar ZnCl 2 . Dashed line box indicates region expanded in C and E. C, top row, selected region of CARD9 -CARD WT 15 N HSQC spectra apo or with equimolar CaCl 2 , MgCl 2 , NiCl 2 , MnCl 2 , or CuCl 2 . Bottom row, selected region of CARD9 -CARD WT 15 N HSQC spectra apo or with equimolar ZnCl 2 , equimolar ZnCl 2 and MnCl 2 , or equimolar ZnCl 2 and CuCl 2 . Arrowheads indicate examples of peak positions unique to the Zn 2ϩ -bound state. D, lowest energy CARD9 -CARD structure with the potential Zn 2ϩ -coordinating residues highlighted in cyan. E, selected region of CARD9 -CARD WT and CARD9 -CARD C37A 15 N HSQC spectra with 1:0 (black), 1:0.5 (green), and 1:1 (red) ZnCl 2 , demonstrating slow exchange dynamics. CARD9 -CARD C10A and CARD9 -CARD H73A are shown with 1:0 (black), 1:0.5 (green), 1:1 (red), 1:2 (orange), and 1:4 (blue) ZnCl 2 , demonstrating a shift to the intermediate-to-fast exchange regime.

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
the positions of which are depicted in Fig. 1D. Although CARD C37A continues to bind Zn 2ϩ in the slow-exchange limit, both CARD C10A and CARD H73A shift binding to the intermediate-to-fast exchange regime, indicating a significant reduction in affinity and demonstrating that Cys-10 and His-73 are involved in coordinating Zn 2ϩ (Fig. 1E). The only glutamates or aspartates potentially in position to provide additional Zn 2ϩ coordination are a stretch of acidic residues (Glu-5, Asp-7, Asp-8, and Glu-9) on the unstructured N-terminal region of the CARD9 -CARD. Upon mutation of Glu-5 or Glu-9 to Gln, we found no substantial differences in the Zn 2ϩ -bound spectrum, indicating no contribution to coordination. In contrast, we found that the Zn 2ϩ -bound spectrum of CARD D7N differs substantially from CARD WT , despite the binding remaining in the slow-exchange regime (Fig. S2). These data suggest that Asp-7 is less critical than Cys-10 or His-73 for Zn 2ϩ affinity but that coordination by Asp-7 results in conformational changes to the CARD. CARD D8N exhibits subtler Zn 2ϩ -bound 15 N HSQC differences as compared with CARD WT , comprising reduced linebroadening of the binding-site-proximal amide peaks Cys-10, Trp-11 (backbone and side chain), Ser-12, and Gln-69 (side chain), suggesting a role for Asp-8 in coordination as well (Fig. S2).

The CARD9 -CARD exhibits a picomolar dissociation constant for Zn 2؉
Zn 2ϩ binding to the CARD9 -CARD is sufficiently tight to preclude direct affinity determination by NMR titration. We instead utilized competition against the fluorescent Zn 2ϩbinding dye mag-fura-2, which has a dissociation constant of 20 nM for Zn 2ϩ (27). As shown in Fig. 2A, the CARD9 -CARD competes effectively against mag-fura-2, although less effectively than EDTA (K D of ϳ10 Ϫ16 for Zn 2ϩ ). Fitting these data to the exact competitive binding equation described by Wang (28), CARD WT binds Zn 2ϩ with a dissociation constant of 0.73 nM (95% confidence interval (CI) 0.45-1.07 nM). Consistent with the NMR data, CARD C10A and CARD H73A are unable to compete with mag-fura-2, whereas CARD C37A binds with comparable affinity to CARD WT . The mutations D7N and D8N each decrease binding affinity ϳ2-3-fold, whereas the double mutant CARD D7N/D8N exhibits an ϳ25-fold decrease, suggesting that the two acidic residues may trade-off responsibility for coordinating the Zn 2ϩ ion (Fig. 2C). Because the measured picomolar affinity of the CARD9 -CARD is approaching the lower limit accessible in competition with the 20 nM magfura-2, we additionally assessed affinity among the tightly binding constructs in competition with the more tightly binding indo-1 dye (K D for Zn 2ϩ of 0.16 nM (29)). As shown in Fig. 2, B and C, affinities as measured in competition with indo-1 agree with those determined with mag-fura-2, confirming that the CARD9 -CARD binds Zn 2ϩ with a picomolar dissociation constant, comparable with estimates of the free cytosolic Zn 2ϩ concentration.

Zn 2؉ binding does not significantly alter the CARD9 -CARD structure
Given the high affinity and specificity that the CARD9 -CARD exhibits for Zn 2ϩ , we were curious as to the impact of Zn 2ϩ binding on the CARD structure. We thus determined near-complete backbone and side-chain chemical shift assignments for the Zn 2ϩ -bound CARD9 -CARD and calculated the NMR solution structure to a backbone RMSD value of 0.5 Å (RMSD calculated for structured residues 10 -97, see Table 1 for complete statistics).
In the Zn 2ϩ -bound structure, the Zn 2ϩ ion forms a bridge between Cys-10 at the beginning of ␣1 and His-73 at the beginning of ␣5 (Fig. 3A). Because of the ambiguity in coordination by Asp-7 and Asp-8, we only imposed constraints to maintain coordination by Cys-10 and His-73 during structure calculations. Although full quantum mechanical calculations were not performed, Asp-7 interacts with the Zn 2ϩ ion with one or both carboxyl oxygens in all of the 20 lowest energy structures, consistent with its prominent role in coordination (representative structures shown in Fig. 3A). The apo and Zn 2ϩ -bound struc- Error bars represent the standard deviation of three technical replicates. Solid lines represent best fits to the generalized competition equation described by Wang (28). C, dissociation constants determined via competition against either mag-fura-2 or indo-1 as indicated. Error bars represent asymmetric profile likelihood 95% CI. For H73A and C10A, no upper limit was found for the 95% CI. For EDTA, either in competition with mag-fura-2 or indo-1, no lower limit was found for the 95% CI.

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
tures are remarkably similar, with a backbone RMSD (residues 10 -97) of 0.95 Å, which is only slightly higher than the RMSD of the apo state itself (Fig. 3B). We further manually compared the NOESY spectra of the CARD in the apo and Zn 2ϩ -bound states in search of more subtle differences in the structures. Although the specific NOE cross-peaks used in the two calculations vary somewhat due to differential peak overlap and line-broadening between the apo and Zn 2ϩ -bound states, we were unable to conclusively identify any instances in which an NOE cross-peak was present in one state and not the other. We therefore conclude that Zn 2ϩ binding does not significantly alter the ground-state solution structure of the CARD9 -CARD.

Zn 2؉ binding stabilizes the CARD9 -CARD and inhibits ␣-helical unraveling
Although binding of a Zn 2ϩ ion to the CARD9 -CARD does not substantially alter its ground-state structure, we were curious as to the potential impact of Zn 2ϩ binding on the CARD stability and conformational dynamics. We monitored denaturation of the CARD9 -CARD monomer via differential scanning fluorimetry and found that addition of Zn 2ϩ increases the thermostability of the CARD by nearly 14°C, reflecting a substantial stabilization of the domain upon Zn 2ϩ binding (Fig. 4A).
To monitor conformational stability, we performed an NMR-based hydrogen-deuterium exchange (HDX) experiment to monitor the solvent accessibility of backbone amides in the apo and Zn 2ϩ -bound states. Aqueous 15 N-labeled CARD9 -CARD was lyophilized and then resuspended in 99.99% D 2 O, followed by a collection of a series of SOFAST-HMQC experiments, which allow for rapid data collection. Approximately 35% of residues remain at least partially protonated by the first 1.5-min time point. For all residues that we were able to monitor, the HDX lifetime was significantly increased in the context of Zn 2ϩ binding, with half-lives increasing by 1.5-14 -fold over the apo state (Fig. 4, B and D). The residues most strongly protected by Zn 2ϩ binding map predominantly to helices ␣4 and ␣5, which lie on either side of His-73 (Fig. 4C). These data demonstrate that Zn 2ϩ binding locks the CARD in a more stable compact conformation, with less conformational breathing in the helices than in the apo state.

The CARD9 -CARD can adopt a domain-swapped dimeric structure
Upon recombinant overexpression of the CARD9 -CARD in E. coli and subsequent purification, two distinct species can be  Two technical replicates are shown for each condition, which agreed to within 0.1°C of the mean values shown on the graph. B, representative NMR HDX peak-height decay curve for Thr-31 in the absence (black) and presence (red) of equimolar Zn 2ϩ . Circles represent 15 N-SOFAST-HMQC peak intensities, and dotted lines are best-fit single exponential decay curves. C, CARD9 -CARD, lowest energy Zn 2ϩ -bound solution structure. Residues for which Zn 2ϩ binding increased the HDX lifetime, which includes all observed peaks, are colored cyan. The indole amide of Trp-11 also exhibits enhanced protection and is colored cyan. Those residues for which lifetimes could be calculated in both the apo and Zn 2ϩ -bound states and were increased greater than 5-fold by Zn 2ϩ binding are colored red. D, global CARD9 -CARD backbone amide HDX exchange lifetimes in the absence (black) and presence (red) of equimolar ZnCl 2 . Error bars represent profile likelihood 95% confidence intervals. Residues are excluded for which no signal remained at the first time point or for which overlap precluded accurate peak height determination.

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
isolated: the monomeric CARD for which NMR structures are shown in Fig. 4, and a kinetically stable dimeric state that accounts for ϳ20% of the purified protein at the final gel filtration step (Fig. 5A). We determined the 1.36-Å resolution X-ray crystal structure of this CARD9 -CARD dimer (see Table 2 for complete statistics) using molecular replacement with the CARD11-CARD structure (PDB code 4LWD). The dimer is composed of two six-helix bundles, each of which aligns well to the CARD9 -CARD monomer (Fig. S3A). Each bundle, however, contains three helices from each of the two polypeptide chains, forming a domain-swapped dimer with a short linker crossing over between helices ␣3 and ␣4 (Figs. 5B and Fig. S3B).
The Zn 2ϩ -binding site in the CARD9 -CARD is distal from the strand swap between ␣3 and ␣4, such that the domain swap would not be expected to alter Zn 2ϩ binding. Consistent with this expectation, NMR chemical shift changes upon addition of Zn 2ϩ to the dimer are nearly identical to those seen for the monomeric state. We thus soaked Zn 2ϩ into domain-swapped dimer crystals, identified a single condition where Zn 2ϩ occupies one of the two binding sites, and solved the crystal structure at a resolution of 1.81 Å (see Table 2 for complete statistics). The Zn 2ϩ ion binds where expected based on our NMR structure of the Zn 2ϩ -bound monomer, with clear electron density demonstrating its coordination by both Cys-10 and His-73 ( Fig. 5C and Fig. S3B). Additional electron density is present, which suggests Zn 2ϩ coordination by a third residue in the N-terminal tail; however, the residues N-terminal of Asp-9

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
are too poorly resolved to conclusively identify which (likely Asp-7 or Asp-8) is participating in the coordination, likely reflecting conformational heterogeneity in the coordination throughout the crystal. In agreement with the minimal groundstate differences observed between the apo and Zn 2ϩ -bound monomeric structures, no notable structural changes are observed in the domain-swapped dimer upon Zn 2ϩ binding (Fig. 5C).
The domain-swapped dimer exhibits a similar 15 N HSQC spectrum as the monomer, but with distinct differences, especially for those residues near the strand swap. By monitoring a single amide peak (Ser-28, Fig. 5D) with resolvable monomeric and dimeric chemical shifts, we were able to track the kinetics of interconversion of the two states. At 25°C, we found that the dimer and monomer interconvert with a half-life of 4.1 h (95% CI 3.8 -5.4 h) in the absence of Zn 2ϩ . This interconversion is dramatically slowed, however, in the presence of Zn 2ϩ , where the dimer exhibits a half-life of 179 h (95% CI 155-206 h), representing a nearly 50-fold decrease in interconversion rate (Fig.  5E). There additionally appears to be a shift in the monomerdimer equilibrium, with ϳ3-fold more dimer present at equilibrium in the presence of Zn 2ϩ relative to the apo state. These findings are consistent with the HDX data, suggesting that the presence of Zn 2ϩ locks the CARD9 -CARD in a more stable conformation, preventing helical unraveling that must be required for the monomeric and domain-swapped dimeric conformations to interconvert.
We observed that the homologous CARD11-CARD (50% identity to the CARD9 -CARD) is also capable of adopting a relatively long-lived dimeric conformation upon overexpression in E. coli, with an in vitro half-life of 34 min (95% CI 28 -48 min) at 25°C (Fig. S4, A-C). We were unable to conclusively demonstrate that the CARD11-CARD adopts a homologous domain-swapped dimeric structure as the CARD9 -CARD, but the long half-life and a comparable extent of chemical shift changes intimate a similar structure. Upon addition of stoichiometric ZnCl 2 to the 15 N-labeled CARD11-CARD monomer, we observed no significant NMR chemical shift perturbations, indicating that Zn 2ϩ binding is not conserved within the protein family (Fig. S4D).

The CARD9 -CARD forms in vitro filaments in a Zn 2؉ -regulated manner
In the absence of Zn 2ϩ , we found that upon concentrating monomeric CARD9 -CARD above ϳ150 M, the solution became cloudy. We visualized this opaque solution by NS-EM and observed that the CARD9 -CARD monomer assembles into long filaments with a diameter of ϳ90 Å (Fig. 6A). To monitor the effect of Zn 2ϩ binding on these filaments, we purified the CARD9 -CARD monomer bound to a stoichiometric amount of Zn 2ϩ by adding a saturating concentration of ZnCl 2 prior to a final gel-filtration column. In contrast to the apo CARD, we found that at 200 M the Zn 2ϩ -bound CARD remained clear by eye and filament-free as monitored by NS-EM (Fig. 6B). Addition of EDTA to chelate the Zn 2ϩ away from the CARDs leads to formation of filaments within ϳ10 min at 25°C. We monitored filament formation though UV absorbance at 350 nm and found that they form readily at 200 and 150 M, but minimally at 100 M (Fig. 6C). Doubling the salt concentration to 300 mM also effectively blocks CARD9 -CARD polymerization at these protein concentrations, a property that permitted NMR data collection and structure determination of the apo CARD9 -CARD monomer described above (Fig. 1A). These assemblies are readily reversible upon re-binding of Zn 2ϩ , as addition of Zn 2ϩ stoichiometrically equal to the EDTA concentration induces disassembly within ϳ5 min. Unlike the monomeric CARD9 -CARD, the domain-swapped CARD9 -CARD dimer solution remains clear at concentrations of Ͼ2 mM, irrespective of the presence of Zn 2ϩ , indicating that the dimeric state of the CARD is unable to form filaments.
We found that Zn 2ϩ binding inhibits filament assembly of the CARD9 -CARD monomer but does not block it entirely. Upon concentrating the Zn 2ϩ -bound CARD9 -CARD solution to ϳ800 M, it also becomes cloudy, and filaments can be observed by NS-EM (Fig. S5A). These filaments are ϳ180 Å in diameter and appear to be tandem assemblies of two filaments, as they often end ϳ90 Å wide, off-center "tails." After addition of Zn 2ϩ to the single-width filaments induced by Zn 2ϩ depletion (as depicted in Fig. 6, A and C), we found that all singlewidth filaments had disassembled when visualized by NS-EM, whereas a small population (undetectable by UV absorbance, Fig. 6C) of tandem filaments had formed, presumably from a subset of filaments that had been able to bind Zn 2ϩ and adopt a stabilized tandem conformation prior to disassembly (Fig.  S5A). In the context of the full CARD9 protein, the coiled-coil domain would necessarily protrude from any helical CARD assembly, likely blocking side-mediated interactions of the filaments. We therefore anticipate that these observed tandem CARD9 -CARD filaments are likely an in vitro artifact. Nonetheless, the vast majority of filaments rapidly disassemble in the presence of Zn 2ϩ , demonstrating that Zn 2ϩ binding regulates the stability of CARD9 -CARD helical assemblies.

CARD9 -CARD filaments comprise a similar helical assembly as Bcl10 and are able to template Bcl10 nucleation
Because CARD9 is thought to propagate signaling via nucleation of Bcl10 helical assemblies, we wondered whether these in vitro filaments are representative of the helical template that seeds Bcl10 polymerization. To determine whether the filaments adopt a conformation consistent with this nucleating capacity, we determined an ϳ20-Å resolution NS-EM structure of the CARD9 -CARD filaments (Fig. 6D, left). The CARD9 -CARD filaments are 90 Å in diameter and form a hollow helical assembly with a 5-Å rise and 102°rotation, which are nearly identical to the 5.0-Å rise and 100.8°rotation determined previously for the Bcl10 -CARD helical assembly (30). Direct comparison of low-resolution NS-EM structures of the CARD9 -CARD and Bcl10 -CARD assemblies (Fig. 6D, right) (EMD-5729 (23)) demonstrates that although the slight differences in helical symmetry lead to discernable differences over several turns of the helix, the two CARDs adopt highly similar filamentous structures. Although the Bcl10 CARD contains an extended C-terminal helix that enables unambiguous fitting of the CARD monomers into the filament structure, the CARD9 -CARD contains no such large asymmetry, preventing us from independently placing our CARD monomeric structure con-

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
clusively into the EM density. However, given the similarity of the helical symmetry between the assemblies, we predict that a higher resolution CARD9 -CARD structure would reveal an orientation comparable with the Bcl10 -CARD filament. The CARD9 -CARD filaments are thus consistent in structure with what we would expect in a templating assembly.
To directly monitor the capacity for the CARD9 -CARD filaments to nucleate Bcl10, we adapted a fluorescence polarization (FP)-based Bcl10 nucleation assay described by Qiao et al. (23). Briefly, a Bcl10 construct was generated linked N-terminally to MBP via a TEV protease-cleavable linker; this MBP tag was shown to block in vitro Bcl10 polymerization, which oth-erwise occurs rapidly for either the full-length Bcl10 or the Bcl10 CARD alone. Bcl10 was also sparsely labeled with Alexa Fluor 488 dye prior to a final gel-filtration column. Bcl10 polymerization is induced by addition of TEV protease, which removes Ͼ50% of the MBP in under 2 min and nearly all MBP within 10 min, irrespective of the presence of CARD9 -CARD (Fig. S5B). Subsequent Bcl10 polymerization is then monitored by the change in FP corresponding to the increased molecular weight of the filament.
As shown in Fig. 6E, addition of CARD9 -CARD filaments to MBP-Bcl10 at a 5:1 molar ratio accelerates the formation of Bcl10 filaments relative to Bcl10 alone, although the addi-

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
tion of monomeric CARD9 -CARD slows Bcl10 polymerization significantly, presumably by competing with Bcl10 homotypic binding sites. We additionally performed a replicate of the experiment utilizing an independent preparation of the CARD9 -CARD filaments and purification of MBP-Bcl10. As shown in Fig. S5C, we observed nearly identical results with an identical replicate (2 M MBP-Bcl10) and additionally found comparable CARD9 -CARD-induced acceleration utilizing a lower concentration of 1 M MBP-Bcl10.
The capacity for the CARD9 -CARD filaments to accelerate bulk Bcl10 polymerization could stem either from direct CARD-CARD templating wherein the Bcl10 -CARD helical assembly extends continuously from the CARD9 -CARD assembly or from indirect effects, e.g. increasing local Bcl10 concentration. To distinguish these possibilities, we visualized CARD9 -CARD filament-nucleated Bcl10 filaments shortly (2 min) after addition of TEV protease by NS-EM. Because Bcl10 contains a C-terminal Ser/Thr-rich domain in addition to its CARD, its filaments are appreciably wider than the CARD9 -CARD filaments (Fig. 6, F and G). As shown in Fig. 6H for the nucleated sample, we were able to readily identify continuous filaments comprising regions of both CARD9 -CARD and Bcl10, demonstrating direct CARD9 -CARD-templated nucleation of the Bcl10 helical assembly. We were unable to find any instance of more than a single CARD9 -Bcl10 transition within a given filament, suggesting that nucleation, like Bcl10 filament extension (30), is unidirectional.

Discussion
Although total eukaryotic cellular Zn 2ϩ concentrations are typically hundreds of micromolar, the vast majority of it is sequestered by tight interactions with proteins (31). Indeed, ϳ10% of the human proteome has been estimated to bind Zn 2ϩ in structural, catalytic, or regulatory capacities (32). Considerable effort has been made to address the challenging question of what concentration of free Zn 2ϩ is readily available in the cytosol of eukaryotic cells, with estimates ranging from 5 pM to 1.67 nM (33,34); however, over diverse cell types and detection methods, most studies have measured concentrations in the range of 0.1-1 nM (35-40). Structural binding sites (e.g. zinc finger domains) typically bind Zn 2ϩ with dissociation constants much lower than the free or readily available cytosolic Zn 2ϩ concentration, and therefore, they remain saturated under all conditions. In contrast, regulatory binding sites typically bind with dissociation constants comparable with the picomolar cytosolic Zn 2ϩ concentration, such that fluctuations in local Zn 2ϩ concentration can modulate function via changes to the Zn 2ϩ -bound status of a given protein (31).
The immune system modulates Zn 2ϩ concentrations from the organismal to the subcellular levels over time scales of seconds to hours in response to diverse stimuli (21,22,41). Within immune cells, several stimuli have been shown to induce a Zn 2ϩ -wave wherein cellular stores of Zn 2ϩ are released on the order of minutes after signal initiation, acting as a second messenger; these include stimulation of IgE in mast cells that activate MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) signaling (20) and activation of monocytes by a range of stimuli, including lipopolysaccharide, inducing downstream NF-B and MAPK signaling (19). Generally, the mechanism of Zn 2ϩ release and the targets of increased cytosolic Zn 2ϩ remain unknown. Specific to CARD9 signaling, Wang et al. (18) demonstrated that Zn 2ϩ deficiency exacerbates cardiac hypertrophy in response to diet-induced obesity and that the onset of disease and an observed Zn 2ϩmediated rescue depend on the activation of p38 MAPK by the CARD9 -Bcl10 signaling axis.
Here, we have demonstrated that the CARD9 -CARD specifically binds to Zn 2ϩ with picomolar affinity, akin to proteins that utilize Zn 2ϩ in a regulatory role; the in vitro affinity suggests a regulatory role for Zn 2ϩ binding to CARD9; however, further characterization will be required to determine to what extent the cellular context influences the binding of Zn 2ϩ to the CARD9 -CARD. To our knowledge, this is the first observation of metal binding in the CARD family or the larger death-domain family. Among the close paralogues to CARD9 (CARD11, CARD10, and CARD14), the coordinating cysteine and histidine are not conserved (Fig. S7A), consistent with the lack of Zn 2ϩ binding observed for the CARD11-CARD (Fig. S4D). The histidine and cysteine are conserved within CARD9 orthologues among mammals but not in reptiles or more divergent species (Fig. S7B).
Although Zn 2ϩ binding does not substantially alter the CARD9 -CARD structure, it does increase its stability, reducing the conformational flexibility of the domain. Moreover, Zn 2ϩ dramatically affects the polymerization propensity of the CARD9 -CARD. Indeed, the most striking in vitro impact of Zn 2ϩ binding that we observed was the modulation of filament formation, shown in Fig. 6, A-C. Although it has been assumed that CARD9, like CARD11, propagates signaling by forming a nucleating seed, Fig. 6 represents the first direct evidence that the CARD of CARD9 is capable of forming a helical assembly, that this assembly closely mirrors the symmetry of the Bcl10 helical assembly, and that the CARD is capable of directly templating Bcl10 polymerization. The micrometer length filaments shown in Fig. 6A were generated with high concentrations of a CARD9 construct lacking the coiled-coil region and are thus unlikely to reflect the size of CARD9 assemblies that would form in cells. Rather, we suggest a model wherein CARD9 -CARDs are brought to a high local concentration by coiled-coil domain-mediated oligomerization, driving formation of a CARD9 -CARD helical "seed" that acts to template and thereby nucleate assembly of Bcl10 filaments.
Given the role of Zn 2ϩ in modulating this template formation, we predict that the primary role of Zn 2ϩ binding in CARD9 will prove to be in modulating the propensity to form this template and hence propagate signaling. Within dendritic cells where CARD9 functions, cytosolic zinc concentrations have been shown to decrease during maturation, potentially serving to prime the CARD9 -CARD for subsequent signaling events (42). Alternatively, a "Zn 2ϩ -wave" type of transient Zn 2ϩ increase could serve to blunt excessive signaling though CARD9 by promoting helical disassembly after stimuli. A detailed temporal study of zinc levels during maturation and stimulation will be required to tease out the specific mechanisms by which Zn 2ϩ binding may modulate the CARD9 signaling axis.

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
The similarity between the NMR solution structures of the apo and Zn 2ϩ -bound CARD9 -CARD (Fig. 3) along with the dramatic differences in stability and conformational flexibility (Fig. 4) suggest that the CARD within the helical assembly may require conformational rearrangement as compared with the monomer in solution. This phenomenon was recently demonstrated for the Bcl10 CARD, for which significant structural rearrangement was observed in a 4-Å resolution cryo-EM structure of the helix as compared with the monomeric NMR solution structure (30). Indeed, the largest differences between the Bcl10 -CARD structures are in the orientation of helix ␣1, which in the CARD9 -CARD contains Cys-10 and would thus be conformationally restricted by Zn 2ϩ binding. Unfortunately, as was reported for filaments of the Bcl10 CARD alone, CARD9 -CARD filaments present primarily as single filaments on NS-EM grids but almost exclusively as massive bundles of filaments (approximately micrometer diameter) when imaging by cryo-EM is attempted. The presence of these large bundles may also help to explain the relatively weak bulk solution nucleating capacity of the CARD9 -CARD filaments (Fig. 6E), as they necessarily sequester large numbers of unproductive CARDs. David et al. (30) were ultimately able to determine a cryo-EM structure of the Bcl10 -CARD filament using a construct containing the Ser/Thr-rich domain that is disordered relative to the CARD core and therefore absent in the reconstruction. Nevertheless, the Ser/Thr-rich domains serve to block side-toside filament associations and are observable en masse on each individual filament, allowing us to distinguish the Bcl10 filaments from the thinner CARD9 -CARD filaments (30). A similar strategy in CARD9 may allow for future high-resolution structure determination, which would provide insight into both homotypic and heterotypic CARD-CARD interactions, as well as into the specific mechanism by which Zn 2ϩ binding modulates CARD9 -CARD helical assembly.
As is common in the death-domain family, the CARD9 -CARD engages in interactions with other CARDs (Fig. 6, D and  H), generating helical assemblies in which the individual domains share a common orientation relative to the helical axis (43). The symmetric nature of the domain-swapped dimer would interfere with this assembly, explaining why the dimer is unable to incorporate into filaments. We thus speculate that the domain-swapped CARD9 -CARD dimer (Fig. 5) may act as a negative regulator of CARD9 signaling that could be modulated by Zn 2ϩ binding. The domain-swapped structure, however, is formed between CARD9 -CARDs under the high concentrations of E. coli overexpression and outside of the context of the full proteins. Further characterization of the full-length protein under physiological conditions, perhaps by utilizing conformationally specific antibodies or an engineered protein deficient in domain swapping, will be required to determine whether the domain-swapped conformation is indeed biologically relevant.
In conclusion, we have identified and structurally characterized multiple conformations accessible to the CARD9 -CARD, including a monomer, a domain-swapped dimer, and a filamentous helical assembly (graphically summarized in Fig. 7). CARD9 binds to Zn 2ϩ with picomolar affinity, which modulates interconversion between these states, stabilizing the CARD9 -CARD ground-state conformation and restricting its capacity to form Bcl10-nucleating filaments. We have thus identified CARD9 as a potential target of Zn 2ϩ -mediated signaling during innate immune responses.

Protein purification
CARD9 and CARD11-CARDs (residues 2-97 for CARD9 and 8 -109 for CARD11) were expressed in BL21(DE3) cells with an N-terminal, TEV protease-cleavable His 6 tag. Protein production was achieved by growth for 48 -72 h at 16°C in TB autoinduction media or 15 N autoinduction media for unlabeled and 15 N-labeled protein, respectively (44). 13 C, 15 N-Labeled protein was generated by induction in 13 C, 15 N minimal media with 0.5 mM IPTG for 6 h at 37°C. Proteins were purified by Ni-NTA (Qiagen), overnight cleavage by TEV protease, removal of imidazole by dialysis, and removal of TEV and any remaining uncleaved protein by Ni-NTA. Final purification and separation of monomeric and dimeric species were achieved via a Superdex 75 gel-filtration column (GE Healthcare). For all proteins used in metal-binding experiments, 5 mM EDTA was added prior to the final gel-filtration column. For proteins used in CARD9 -CARD filament assays, 500 M ZnCl 2 was added after the second Ni-NTA column, prior to concentration before the gel-filtration step.
For the Bcl10 FP assay, an E. coli expression construct was generated comprising an N-terminal His 6 tag followed by MBP, a TEV cleavage site, Bcl10 C29A/C10A , a short linker, and an HA peptide (MBP-Bcl10). The cysteines were mutated to allow for labeling exclusively on the Ser/Thr-rich domain and not the CARD. The ORF was designed to exactly replicate the construct generated by Qiao et al. (23), with the addition of GSGSYPYDVPDYA at the C terminus. MBP-Bcl10 was expressed in BL21(DE3) cells in LB media, which were induced by addition of 0.2 mM IPTG at A 600 0.7 for 1 h at 37°C. MBP-Bcl10 was purified by Ni-NTA and eluted in ϳ3 ml, to which 2 M Alexa Fluor 488 C 5 maleimide (ThermoFisher Scientific)

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
was added, followed by incubation for 10 min at room temperature. The labeled protein was then loaded directly with no concentration onto a Superdex 200 gel-filtration column (GE Healthcare) in 20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.5. Both the FP assay and preparation of grids for NS-EM were initiated within 2 h of the protein eluting from the gel-filtration column.

NMR assignments and solution structure determination
For apo assignments and structure determination, monomeric 13 C, 15 N-labeled CARD9 -CARD was purified in 50 mM Tris, 300 mM NaCl, 0.5 mM TCEP, pH 7.0, and concentrated to 400 M for all NMR experiments. All experiments were collected at 37°C on an 800-MHz Bruker spectrometer with a cryogenically cooled probe. Backbone assignments were determined through sequential assignments using 15  For the Zn 2ϩ -bound CARD9 -CARD, monomeric 13 C, 15 Nlabeled CARD was purified as above to 400 M followed by addition of 480 M ZnCl 2 . Backbone and side-chain assignments were transferred from the apo form, utilizing 15 N HSQC, HNCACB, HNCA, 13 C HSQC (aliphatic and aromatic), HCCH-TOCSY, and 13 C NOESY-HSQC (aliphatic and aromatic, 150 ms mixing times) experiments, with additional 15 N NOESY-HSQC (150-ms mixing time) and 13 C NOESY-HSQC (aliphatic and aromatic, 150 ms mixing times, dissolved in 99.99% D 2 O) experiments collected to assist in structure determination. Stereospecific assignments for valine and leucine methyl groups were determined for the Zn 2ϩ -bound CARD9 -CARD by expressing the protein in M9 media with a 1:9 13 C/ 12 C glucose ratio and collection of 13 C HSCQ spectra as described by Senn et al. (45); these stereospecific assignments were subsequently transferred to the spectra of the apo protein.
For both apo and Zn 2ϩ -bound samples, a small proportion of the monomeric protein converted to the domain-swapped dimer over the course of extended NMR data collection; however, the concentration remained sufficiently low as to not register in the 3D experiments and was therefore ignored for both sequential assignments and structure calculation.
For structure determination of both the apo and Zn 2ϩbound CARD9 -CARD, dihedral angles were estimated using TALOSϩ (47). For the Zn 2ϩ -bound CARD, restraints were enforced to maintain coordination by Cys-10 S␥ and His-73 N␦1. NOE peaks were assigned and initial structure determination was achieved using the CYANA version 3.97 NOE assign-ment and structure determination package (48,49). Sum of r Ϫ6 averaging was used for all NOEs. For each round of CYANA NOE assignment and structure determination, 100 structures were generated, with the 20 lowest target function structures proceeding to the next round. After the final round of NOE assignments, 100 structures were calculated and subsequently refined in explicit water using the PARAM19 force field in CNS version 1.2 (50,51) and the WaterRefCNS package developed by Dr. Robert Tejero. The 20 lowest energy structures for each of the apo and Zn 2ϩ -bound refinements in water are presented here. Structures were evaluated using PROCHECK-NMR, with statistics presented in Table 1. All structural depictions were generated in PyMOL. 15

Metal competition assays
Competition assays were performed in 20-l volumes in 10 mM HEPES, 150 mM NaCl, pH 7.5. Wildtype (WT) or mutant CARD9 -CARDs were mixed 1:1 with either mag-fura-2 or indo-1 dyes, with final concentrations of 10 M each. Stock CARD9 -CARD concentrations were determined by measuring absorbance at 280 nm; mag-fura-2 and indo-1 stock concentrations were determined by measuring absorbance at 369 and 346 nm, respectively, in the presence of EDTA. CARD9 -CARD and dye were incubated at 25°C with the indicated concentrations of Zn 2ϩ for 15 and 45 min for mag-fura-2 and indo-1, respectively, to ensure that measurements were made under equilibrium conditions. For mag-fura-2 samples, Zn 2ϩ binding was monitored by measuring emission at 497 nm upon excitation at 325 nm. For indo-1 samples, Zn 2ϩ binding was monitored by measuring emission at 460 nm upon excitation at 320 nm. All measurements were made on a Molecular Devices SpectraMax M5e plate reader. Data were fit to the exact competitive binding equation described by Wang (28) using GraphPad Prism 7.

Differential scanning fluorimetry
Protein denaturation was monitored by differential scanning fluorimetry using the NanoTemper Prometheus NT.48 for both data collection and analysis. 100 M CARD9 -CARD was prepared with or without addition of 100 M ZnCl 2 in 50 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 7.5. Temperature was increased at 1°C/min, and the protein-folding state was monitored via the ratio of tryptophan fluorescence at 350 and 330 nm. Melting temperature was determined as the inflection point of the 350/330 ratio.

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
Hydrogen-deuterium exchange 250 M 15 N-labeled monomeric CARD9 -CARD samples were generated in 50 mM HEPES, 300 mM NaCl, 250 M ZnCl 2 , 0.5 mM TCEP, pH 7.0, with or without 1 mM EDTA. Samples were lyophilized and resuspended in 99.99% D 2 O, followed by immediate collection of 15 N-labeled SOFAST-HMQC (52) experiments at 25°C on a Bruker 600-MHz spectrometer for time points shown. The mid-point of the SOFAST-HMQC experiments was used as the time points for both plotting and fitting. For all resolved peaks, peak height decay curves were fit to a single-phase exponential using GraphPad Prism 7.

Crystallography and structure determination
Dimeric WT CARD9 -CARD was purified in 20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.0, and concentrated for crystallization. Apo crystals were generated by vapor diffusion at 19°C in 0.4-l sitting drops by mixing 20 mg/ml CARD9 -CARD dimer and 1.1 M ammonium tartrate dibasic, pH 7.0 (Hampton Research), at a 1:1 ratio. Crystals were transferred into a cryoprotectant solution of the crystallization solution supplemented with 20% glycerol and frozen in liquid nitrogen. Crystals into which Zn 2ϩ was soaked were generated by vapor diffusion at 19°C in 0.4-l sitting drops comprising a 1:1 mix of 20 mg/ml CARD9 -CARD dimer and 5% v/v tacsimate, 0.1 M HEPES, pH 7.0, 10% w/v PEG monomethyl ether 5,000 (Hampton Research). These crystals were transferred into the crystallization buffer supplemented with 3 mM ZnCl 2 and incubated ϳ16 h at 19°C. Crystals were then transferred into a cryo-protectant solution of the crystallization solution supplemented with both 1 mM ZnCl 2 and 20% glycerol and subsequently frozen in liquid nitrogen.
Diffraction images were collected at the Advanced Light Source beamline 5.0.2. Data were indexed, integrated, and scaled using XDS and XSCALE (53). Both structures were solved by molecular replacement with Phaser-MR within the Phenix package (54,55), using the monomeric CARD11-CARD structure (PDB code 4LWD) as a search model for the apo structure and the apo domain-swapped dimer structure as a search model for the Zn 2ϩ -bound structure. For both structures, iterative cycles of model building in COOT (56) and refinement in Phenix were used to generate final models. All structural depictions were generated in PyMOL.

Monomer-dimer interconversion kinetics
300 M 15 N-labeled dimeric CARD9 -CARD samples were generated in 50 mM HEPES, 300 mM NaCl, 500 M ZnCl 2 , 0.5 mM TCEP, pH 7.0, with or without 1 mM EDTA. Samples were transferred to 25°C followed by immediate collection of SOFAST-HMQC (52) experiments at 25°C on a Bruker 600-MHz spectrometer. Between time points, samples were incubated at 25°C. For CARD11, 180 M 15 N-labeled dimeric CARD11-CARD was prepared in 50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.0; the sample was generated by concentrating fractions from the dimer peak in Fig. S4A at 4°C for ϳ1 h, followed by immediate data collection at 25°C. The mid-point of the SOFAST-HMQC experiments was used as the time points for both plotting and fitting. Monomeric and dimeric peak heights were fit simultaneously to a single-phase exponen-tial for each sample using GraphPad Prism 7 using amide peak of Ser-28 (CARD9) or the peak boxed out in Fig. S4B (CARD11).

CARD9 -CARD filament formation
Zn 2ϩ -bound CARD9 -CARD was prepared by saturating with Zn 2ϩ prior to a final size-exclusion purification step. 50 l of Zn 2ϩ -bound CARD9 -CARD was prepared in 50 mM Tris, 150 or 300 mM NaCl, 0.5 mM TCEP at the indicated concentrations in a 384-well clear-bottom plate. At t ϭ 0, Zn 2ϩ was removed by addition of 250 M EDTA. Filament formation was monitored by measuring absorbance at 350 nm on a Molecular Devices SpectraMax M5e plate reader while shaking at 25°C. At the indicated time point, an additional 250 M ZnCl 2 was added to each well, and monitoring was continued. Samples for EM were taken just prior to EDTA addition, just prior to Zn 2ϩ addition, and at the end of the assay.

Bcl10 fluorescence polarization assay
The assay was performed in a 20-l volume in 20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.5, with 1 or 2 M final concentration of MBP-Bcl10 as indicated. CARD9 -CARD filaments were prepared as in Fig. 6C by addition of 250 M EDTA to 200 M Zn 2ϩ -saturated CARD9 -CARD, followed by incubation with shaking at 25°C for 90 min. The CARD9 -CARD monomer control was treated identically to the filament sample, but without addition of EDTA. At the initiation of the experiment, TEV protease was added to 0.05 mg/ml along with CARD9 -CARD monomer or filaments as indicated. Fluorescence polarization was measured by exciting at 495 nm and monitoring at 519 nm on a Molecular Devices SpectraMax M5e plate reader at 25°C. We note that by the 90-min end point of the FP assay at 25°C, small numbers of filaments form in the MBP-Bcl10 sample in the absence of TEV, indicating that the MBP is not absolute in its ability to block polymerization (Fig.  S5D). These filaments are sufficiently sparse as to not register on the FP assay (Fig. 6E) and much thicker than the Bcl10 filaments that form upon TEV cleavage, ensuring that we are not observing them in Fig. 6, F and H.

EM and helical reconstruction
Negative stain samples were generated by incubating on glow-discharged carbon on 400-mesh copper grids (Electron Microscopy Sciences) for 30 s, followed by staining with 2% uranyl acetate. For Fig. 6, A and B, and Fig. S5, A and D, and micrographs used for helical reconstruction of the CARD9 -CARD filaments, 4-l samples were applied at 200 M with no dilution. For Fig. 6, F and H, samples were prepared by mixing MBP-Bcl10 (2 M), CARD9 -CARD filaments (10 M), and/or TEV (0.05 mg/ml) as indicated and incubating at room temperature for 2 min followed by direct application of 4 l onto the grid, with no dilution.
Grids were imaged using a Talos F200C microscope operated at 200 kV and Ceta camera (ThermoFisher Scientific). Images for helical reconstruction were collected at 2.006 Å/pixel; all other images were collected at 4.097 Å/pixel. For helical reconstruction of the CARD9 -CARD filament, filaments were manually picked using the EMAN2 program e2helixboxer (57), and

Zinc binding modulates CARD9 -CARD nucleation of Bcl10
all subsequent processing steps were performed utilizing routines in Spring (58). CTF parameters were determined using Micctfdetermine, which utilizes CTFFIND (59). Micrographs were CTF corrected and segmented using Segment, yielding 16,754 segments, which were classified into 50 classes using Segmentclass. Six of these classes (chosen by visual inspection) were analyzed using Segclassreconstruct, which computes a 3D reconstruction based on a single class average over a set of incremented helical symmetries (i.e. rise and rotation); the projections of these reconstructions are quantitatively compared against the original class average to identify helical symmetries compatible with the class average. Of those helical parameters that returned a high-correlation coefficient, the 3D reconstructions were visually inspected to identify structures that contained volumes compatible with the globular CARD9 -CARD. These helical parameters were then used as an input to Segmentrefine3D to iteratively refine the structure from the segment stack, beginning from a cylinder of radius 100 Å. Finally, the 2D projections and power spectra of the reconstructions were compared against the class averages; only the reported parameters of a 5-Å rise and 102°rotation yielded a projection and power spectrum that matched the class averages (Fig. S6, A-C). The final reconstruction utilized 11,226 segments. Using six independent class averages, we ran Segclassreconstruct with fine spacing (0.1 Å and 0.1°) around 5 Å and 102°. The highest correlation symmetry parameters in all cases were within Ϯ 0.1 Å and Ϯ 0.5°of the reported symmetry, providing an estimate of the uncertainty in these values. Fourier shell correlation analysis (Fig. S6D) of the final structure indicates a resolution of 13.0 Å; however, we were unable to discern significant structural details beyond the location of the individual CARD9 -CARDs, and so we suggest by comparison with other comparable structures that the resolution is ϳ20 Å.

Sequence alignment
Multiple sequence alignments were performed using Clustal Omega (60).