Structure and Interdomain Dynamics of Apoptosis-associated Speck-like Protein Containing a CARD (ASC)*

The human protein ASC is a key mediator in apoptosis and inflammation. Through its two death domains (pyrin and CARD) ASC interacts with cell death executioners, acts as an essential adapter for inflammasome integrity, and oligomerizes into functional supramolecular assemblies. However, these functions are not understood at the structural-dynamic level. This study reports the solution structure and interdomain dynamics of full-length ASC. The pyrin and CARD domains are structurally independent six-helix bundle motifs connected by a 23-residue linker. The CARD structure reveals two distinctive characteristics; helix 1 is not fragmented as in all other known CARDs, and its electrostatic surface shows a uniform distribution of positive and negative charges, whereas these are commonly separated into two areas in other death domains. The linker adopts residual structure resulting in a back-to-back orientation of the domains, which avoids steric interference of each domain with the binding site of the other. NMR relaxation experiments show that the linker is flexible despite the residual structure. This flexibility could help expand the relative volume occupied by each domain, thus increasing the capture radius for effectors. Based on the ASC structure, a tentative model is proposed to illustrate how ASC oligomerizes via CARD and pyrin homophilic interactions. Moreover, ASC oligomers have been analyzed by atomic force microscopy, showing a predominant species of disk-like particles of ∼12-nm diameter and ∼1-nm height. Taken together, these results provide structural insight into the behavior of ASC as an adapter molecule.

four family subclasses (1): i.e. death domains (DD), 2 death effector domains (DED), caspase recruitment domains (CARD), and pyrin domains (PYD). Most members of this superfamily are composed of multiple domains, typically from two to six, which mediate homotypic interactions within each domain subfamily (1). In particular, DD/DD, DED/DED and CARD/CARD interactions have been characterized structurally (1), whereas the PYD/PYD binding mode is currently unknown.
The human protein ASC (2, 3) is a member of the death domain superfamily bearing two death domains (N-terminal PYD, C-terminal CARD). ASC functions as an adapter molecule in both apoptosis and inflammation by interacting with Bax (4,5) and caspases (6,7) during apoptosis and by regulating the caspase-1-dependent inflammatory form of cell death named pyroptosis (8). ASC involvement in the cell death machinery seems to be connected to human diseases such as cancer. In fact, ASC gene transcription is impeded by aberrant DNA methylation in numerous types of human cancer (breast, ovarian, brain, and prostate) (9).
Additionally, ASC acts as an integral adapter in the assembly of the inflammasome, a multiprotein complex necessary to activate caspase-1 leading to the processing and secretion of proinflammatory cytokines (10 -12). The inflammasome comprises the NOD/NACHT-LRR proteins, ASC, and caspases 1 and 5 (13). NOD/NACHT-LRR proteins (14) recognize pathogenassociated molecular patterns and lead to a cascade of interactions responsible for caspase-1 activation. This molecular cascade regulates the processing of interleukin-1␤ family members. The several types of inflammasomes identified to date differ in the NOD/NACHT-LRR protein (Ipaf, NALP1, NALP2, cryopyrin/NALP3, and pyrin) and the cellular mechanism followed to activate caspase-1. The NALP2-, cryopyrin/NALP3-, and pyrin-dependent inflammasomes do not associate directly with caspase-1 and require ASC as an adapter (15)(16)(17)(18)(19). This assembly is mediated by homophilic interactions between the PYD and CARD of ASC with the PYD-containing NOD/NACHT-LRR and the CARD of procaspase-1 (13,15,20,21).
In addition, ASC oligomerizes into supramolecular and functional complexes. For instance, inflammatory stimuli in macrophages induce the formation of a large ASC complex named pyroptosome, which is a potent caspase-1 activator responsible for pyroptosis (8). This ASC assembly has been observed in vitro as well (22). During apoptosis, ASC also oligomerizes into structures (2,6) that serve as the scaffold for supramolecular platforms involved in caspase activation (13,18). The multiple functions of ASC have granted it the nickname of "molecular glue" (10).
ASC is an attractive candidate in the death domain superfamily for structural studies because it performs critical roles in apoptosis and inflammation with the simplest molecular architecture consisting of two domains. The structural and dynamic characterization of full-length ASC could help to improve our current understanding of the role played by interdomain conformational dynamics and the interplay between domains in the function of death domain proteins. What is known structurally on this subject emerges from the reported 3D structures of only two proteins with more that one death domain: i.e. FADD (composed of a DD and a DED) (23) and MC159 (a tandem of two DEDs) (24). The structure of FADD shows a tail-to-tail orientation of its two domains that results from the short (six-amino acid) linker connecting them and few contacts between both domains located in the vicinity of the linker. In contrast, the two DEDs of MC159 form a binding interface, as expected from the propensity of death domains to form homotypic interactions within members of the same subfamily (1). To date, the 3D structure of the pyrin domain of ASC is known (25). On the basis of this structure it has been suggested that the PYD/PYD interaction is analogous to the CARD/CARD binding mode (26,27). However, the absence of the CARD in this structure precludes investigating the interplay between domains in ASC function.
This study reports the high resolution NMR structure of fulllength ASC together with the analysis of its interdomain dynamics using NMR relaxation techniques. The results show that the PYD and CARD of ASC are structurally independent and connected by a flexible linker. The linker displays some local structure that restrains interdomain dynamics, leading to a back-to-back orientation of the two domains that facilitates binding to multiple partners. The interdomain flexibility of ASC could operate in a "fly-casting" fashion (28) to increase its ability to capture binding partners. By combining the ASC structure with the single known 3D structure of a CARD/CARD complex (27), a model for ASC dimerization is proposed. The model illustrates that the PYD and CARD of ASC are confined in space without obstructing the binding of each domain to their respective partners and suggests a possible way to oligomerize into larger assemblies. To complement the high resolution structural and dynamics studies by NMR, ASC oligomers have been analyzed by atomic force microscopy showing that ASC oligomerizes into ϳ12-nm-diameter and ϳ1-nm-height disk-like particles. The results reported herein provide structural and dynamic insights into how ASC can operate as molecular glue through protein-protein interactions mediated by its two death domains.

EXPERIMENTAL PROCEDURES
Protein Cloning, Expression, and Purification-Cloning, expression, and purification of human ASC has been reported elsewhere (29).  (30,31). All experiments were processed with NMRPipe (32) and analyzed with PIPP (33). Spectra resulting from some of these experiments are shown in Fig. 1 and supplemental Fig. S1.
Several attempts were made to obtain residual dipolar couplings, including the use of bicelle (34) and bacteriophage (35,36) alignment systems. ASC appears to disrupt bicelle formation resulting in the absence of protein alignment. In the presence of bacteriophages, the NMR spectrum mostly shows signals corresponding to the flexible linker, therefore, precluding the measurement of sufficient data to include in the structure calculation protocol.
Structure Calculation-Peak intensities from NOESY experiments were translated into a continuous distribution of interproton distances. Distances involving methyl groups, aromatic ring protons, and non-stereospecifically assigned methylene protons were represented as a summation averaging, (Αr Ϫ6 ) Ϫ1/6 (37). Errors of 40 and 30% of the distances were applied to obtain lower and upper distance limits. 78 hydrogen bond distance restraints (r NH-O ϭ 1.9 -2.5 Å, r N-O ϭ 2.8 -3.4 Å) were defined according to the experimentally determined secondary structure of the protein. The TALOS program (38) was used to obtain 311 and restraints for those residues with statistically significant predictions. Structures were calculated with the program X-PLOR-NIH 2.16.0 (39). The starting structure was heated to 3000 K and cooled in 30,000 steps of 0.002 ps during simulated annealing. The final ensemble of 20 NMR structures was selected based on lowest energy and no restraint-violation criteria. The 20 lowest energy conformers have no distance restraint violations and no dihedral angle violations greater than 0.35 Å and 4.5°, respectively. Structure quality was assessed with PROCHECK-NMR (40) and MolProbity (41). Structures were analyzed with MOLMOL (42). Coordinates were deposited in the Protein Data Bank with accession code 2KN6.
Backbone 15 N Relaxation Measurements-Relaxation experiments were performed at 298 K in a Bruker Avance spectrometer operating at 600 MHz. The 15 N T 1 , T 1 , and { 1 H}-15 N NOE data were obtained with specific NMR pulse sequences (43,44). The recycle delay to measure 15 N T 1 and T 1 was 1 s, whereas { 1 H}-15 N NOE experiments used 3.2s. All experiments were acquired in an interleaved manner to minimize the effects caused by spectrometer drift. The relaxation delays of T 1 experiments were the following: 12, 36, 100, 244, 484, 964, 1284, and 1604 ms. T 1 experiments used a 15 N continuous spin-lock field of 2.5 kHz. T 1 , instead of T 2 relaxation times were acquired because resonance offset effects are significant in T 2 experiments, whereas they can be corrected in a straightforward manner for T 1 data using the equation (45), where ϭ tan Ϫ1 (⍀ N /␥ N B 1 ), ⍀ N is the resonance offset, and ␥ N B 1 is the strength of the spin-lock field. T 2 values can be obtained from Equation 1, as T 1 , T 1 , and are known.
Relaxation times were calculated by fitting peak-intensity dependence with the experiment relaxation times to an exponential function given by I(t) ϭ I 0 e[(Ϫ1/T)t] (T ϭ T 1 , T 1 ). T 1 and T 1 values are averages of two separate measurements. The { 1 H}-15 N NOE values were calculated from the ratio of peak intensities obtained from experiments performed with and without 1 H presaturation. The 1 H frequency was shifted offresonance in the unsaturated experiments. The pulse train used for 1 H presaturation utilized 162°pulses separated by 50-ms delays and was applied for a total of 2.2s. The recycle time is reasonably long; however, NOE values were corrected for incomplete 1 H magnetization recovery as previously described (44).
Apparent rotational correlation times were obtained assuming full anisotropy, as described elsewhere (46), from relaxation data of residues that do not undergo slow conformational averaging and show { 1 H}-15 N NOE values larger than 0.65 (43). The parameters of the rotational diffusion tensor are shown in supplemental Table S1.
Atomic Force Microscopy Imaging-Mica surfaces were covered with 3 l of protein solution at either pH 3.8 or 7.0 and incubated for 30 s. The surface was subsequently rinsed with the respective buffers at pH 3.8 and 7.0 and dried. Tapping mode imaging in air was conducted with a Multimode Atomic Force Microscope (Veeco Instruments, Santa Barbara, CA) using a Nanoscope IIIa controller and a J scanner. Veeco nanoprobe tips TESP7 with a resonance frequency of 320 kHz and a spring constant k ϭ 42 newtons/m were used. Scan rates were set at 1Hz.
Molecular Modeling-The solution structure of ASC was used as the monomer template to build the model for the ASC dimer by superimposing the CARD of ASC to Apaf-1-CARD and caspase-9-CARD complex (27). The model was created with the program MOLMOL (42).

RESULTS
ASC Propensity to Oligomerize by NMR and AFM-ASC selfassociates in vivo during apoptosis and inflammation (2) and is capable of forming functional supramolecular assemblies in vitro (22). The oligomerization of ASC poses significant challenges for NMR structural studies regarding protein solubility and particle size. It is, therefore, critical to find conditions to minimize oligomerization at the relatively high concentrations used in protein NMR (ϳ1 mM).
ASC solubility is very low at neutral pH. The soluble fraction of ASC at pH 7 cannot be detected with Coomassie Blue staining in polyacrylamide gel electrophoresis (detection limit 50 -100 ng) and results in few observable signals with intensity slightly above the noise level in a spectrum acquired using fast NMR acquisition techniques (47) (supplemental Fig. S2). In contrast, under acidic conditions (pH ϳ 4) ASC solubility improves and can readily be detected by NMR. The dispersion of NMR signals in the [ 1 H, 15 N]-HSQC spectrum of ASC ( Fig.  1A and supplemental Fig. S2) indicates that the protein is properly folded at this pH. However, NMR signal intensity decreases over time with no concomitant changes in the chemical shifts (Fig. 1B). This result suggests that ASC forms oligomers of considerable size that tumble too slowly to be observed in the NMR spectrum. The NMR signal intensity of 0.7 mM ASC decays to ϳ80% after ϳ3 h of sample preparation and to ϳ30% within the first 5 days (Fig. 1B). In contrast, at 0.2 mM protein concentration, signal intensity decays to 98% after ϳ3 h of sample preparation and to ϳ80% within the first 24 h. NMR signal intensity continues decaying to ϳ75% of the original signal, reaching a plateau after ϳ3 days of sample preparation. Therefore, by decreasing the protein concentration to 0.2 mM, the effect of ASC oligomerization in NMR signal intensity is significantly reduced, whereas it is still possible to acquire NMR triple-resonance spectra with a signal enhancing cryogenic probe.
Because the capability of ASC to oligomerize is basic to its function, oligomerization at neutral and acidic pH was investi- gated by scanning atomic force microscopy. AFM images show that ASC forms oligomers of similar size and shape at both pH values (Fig. 2, A and B). The predominant species appears like disks of ϳ1-nm height and ϳ12-nm diameter (see the section images in Fig. 2, A and B). Taken together, the NMR and AFM data indicate that ASC is able to oligomerize in vitro into particles of similar structural features under both pH conditions. Based on these results, it is reasonable to assume that the structure of ASC is not perturbed at acidic pH. Under acidic conditions, the oligomerization reaction likely favors the monomeric form, increasing in turn the solubility of ASC and, thus, leading to a larger fraction of monomers observable by NMR.
To investigate whether the possible oligomerization of ASC at 0.2 mM interferes with structural and dynamics studies, NMR relaxation measurements were performed. Backbone amide ( 15 N) magnetic relaxation experiments provide rotational correlation times ( c ), which directly depend on the molecular size and shape (46,48). In the presence of aggregation, measured c values should be larger than theoretical values derived from the molecular size (49). Differences between the two can also originate from the low sphericity of the protein and from dynamic processes associated, for example, to interdomain motion. The former case generally results in experimental c values larger than the prediction. The experimental c value of ASC is small for its size (ϳ22 kDa) ( Table  1), indicating that the fraction of ASC molecules observable by NMR tumbles as monomers. The discrepancy with the theoretical value (Table 1) could, therefore, be due to interdomain dynamics in ASC (see below). In addition, amide 15 N transverse relaxation times (T 2 ) depend on the protein rotational correlation time but are reduced in the presence of aggregation (50). For ASC, the average T 2 value of the two domains (72.5 Ϯ 5.5 ms) agrees with the measured correlation time.
These results indicate that ASC oligomerization does not have significant effects under the conditions used for the following NMR structural and dynamics studies.
High Resolution Structure of Full-length Human ASC-The three-dimensional structure of ASC was determined with 3046 NOE-derived distances and 311 dihedral and 78 hydrogen bond restraints. The 20 lowest energy conformers of ASC do not show distance or angle restraint violations greater than 0.35 Å and 4.5°, respectively ( Table 2). The ensemble of structures does not show significant deviations from covalent geometry and is well defined by the NMR data, resulting in low atomic coordinate precision for the backbone and all heavy atoms ( Table 2). Structural validation data of the ASC structure obtained with MolProbity (41) in comparison to average values calculated for all NMR PYD and CARD structures deposited in the Protein Data Bank indicate that the structure of ASC is of comparable quality ( Table 3). The equivalent resolution provided by PROCHECK-NMR (40) of the ASC structure compared with x-ray structures is between 1 and 1.8 Å (supplemental Fig. S3).  The NMR structure of full-length ASC shows two six-helix bundle domains (PYD and CARD) connected by a 23-residuelong linker (Fig. 3A). No interdomain NMR-derived contacts (NOEs) were observed, and neither domain shows NOEs with the linker; therefore, the PYD and CARD do not interact with each other. This result agrees with the previously observed propensity of death domains to participate in homotypic interactions within each subfamily (1) and the description of ASC as an adapter protein with two homophilic protein-protein interacting domains (51). Although long, the linker of ASC (residues 90 -112) adopts some residual structure as evidenced by the presence of short-range NOEs. In addition, the 13 C ␣ chemical shifts (29) deviate from random coil values. NOE data involving residues 90 -94 (supplemental Table S2) and some positive 13 C ␣ secondary shifts in this region (Fig. 4A) suggest that it adopts residual turn-type conformations. In contrast, 13 C ␣ chemical shift deviations are almost consistently negative from residues 95 to 112 (Fig. 4A), pointing to the formation of low populated extended structures (52). These results are supported by the empirical program TALOS (38), which using a combination of ASC chemical shifts of several nuclei ( 15 N amide, 13 C ␣ , 13 C ␤ , 13 CЈ, 1 H ␣ ) as input data, predicts most linker residues to populate extended structures (Fig. 4B). It is noteworthy that only five residues fall outside the extended structure region of the Ramachandran plot. Three of them (Gln-91, Gly-92, and Gly-94) belong to the fragment 90 -94 that, based on NOE data, is likely adopting turn or short-helix conformations. The other two residues (Gly-99 and Gly-111) are comprised in the fragment 95-112 and fall in the left-handed helix region characteristic of Gly. The propensity of the linker to adopt extended structure is not surprising on the basis of its amino acid composition. Residues such as Ser, Ala, Gly, and Pro, present in the linker of ASC, are known to bias the polypeptide chain toward such conformations (53)(54). In particular, two consecutive Pro residues (Pro-103 and Pro-104 in the linker of ASC) favor the polyproline II or collagen conformation (54), which is a common residual extended structure found in protein loops and linkers connecting domains in modular proteins (53).
In contrast to the structure of full-length ASC reported here, in the solution structure of the protein FADD, few interdomain NMR contacts have been observed between the DD and the DED (23). These interactions are not located in the consensus binding sites of each domain and are spatially close to the short linker (6 amino acids) connecting them. The linker length and flexibility could, thus, emerge as important factors in the structure and dynamics of the death domain superfamily.
The PYD of full-length ASC shares some general characteristics with other known PYD structures, including the long loop between helices 2 and 3 (Figs. 3A and 4), a feature of PYDs absent in other death domains. However, it is worth noting several differences with the isolated PYDs of NALP1 (55) and NALP10 (PDB entry 2DO9). These show a disordered loop in place of helix 3 in the PYD of ASC (Fig. 3B). Helices 1 and 6 are also shorter in the PYD of NALP1 (Fig. 3B). These structural differences result in high root mean square deviation values (9.7 Å for NALP1-PYD and 7.3 Å for NALP10-PYD) and might be related to their differences in biological function. In fact,   NALPs contain additional domains and leucine-rich motifs, suggesting different roles in inflammasome formation. In contrast, the PYD-only protein ASC2 (56), which is thought to modulate ASC-mediated autoimmune response through PYD/ PYD interactions (57), is significantly similar to the PYD of ASC at the structural level (root mean square deviation ϭ 1.36 Å) and also displays the helix 3. Not surprisingly, the PYDs of both proteins share a high degree of sequence identity (64%). The PYD of full-length ASC and the isolated PYD (25) are structurally very similar as well (root mean square deviation ϭ 1.37 Å). This result indicates that the presence of the CARD does not perturb the structure of the PYD in ASC and agrees with the absence of interdomain contacts.
Full-length ASC CARD shows structural peculiarities compared with other known CARDs. In all previously reported CARD structures, helix 1 is bent or broken into two smaller   (55). Right panel, shown are full-length ASC PYD (red) and NALP10-PYD (orange) (PDB entry 2DO9). C, shown is the superposition of the structures of full-length ASC CARD (green) and Apaf-1-CARD (navy) (26). Helices are shown as cylinders in B and C. D, electrostatic surface of full-length ASC CARD and Apaf-1-CARD (only the negatively charged area is shown in Apaf-1-CARD). Protein orientation is equivalent. NOVEMBER 20, 2009 • VOLUME 284 • NUMBER 47 helices, named H1a and H1b. The hinge connecting these two fragments is involved in protein-protein interactions according to structural studies on the complex between Apaf-1-CARD and caspase-9-CARD (26,27). Fragment H1a is missing in the CARD of ASC. Helix 1 spans residues Gln-117 to Val-126 and is preceded by a relatively ordered turn (His-113-Asp-116) (Fig.  3A). A comparison of the CARD structure of full-length ASC with the solution structure of Apaf-1-CARD (26), which is one example with the two H1a and H1b fragments, is shown in Fig.  3C. The H1a fragment displayed by Apaf-1-CARD corresponds to residues 108 -116 in ASC. The region 108 -112 is significantly flexible according to NMR relaxation data (see below), thus confirming the absence of H1a in ASC. The lack of H1a in the CARD of ASC might be related to its plasticity in proteinprotein interactions that could facilitate participation in apoptotic and inflammatory events. In addition, large deviations in the orientation of helices 2 and 3 are also observed (Fig. 3C), which can result from the propagation of structural changes in the binding surface involving the connection between H1a and H1b. The electrostatic surface of full-length ASC CARD is significantly different from Apaf-1 CARD (Fig. 3D). ASC shows positively and negatively charged areas evenly spread throughout the surface, whereas Apaf-1-CARD shows two extensive oppositely charged patches (Fig. 3D). Within a similar fold, CARDs show structural differences pertaining to helix length, orientation (1), and electrostatic surface (examples are illustrated in supplemental Fig. S4), which might serve as a finetuned mechanism to tightly control the binding specificity observed in protein-protein interactions mediated by these domains.

Structure and Interdomain Dynamics of ASC
Interdomain Dynamics in ASC-NMR relaxation of backbone amide ( 15 N) measured as heteronuclear Overhauser values ({ 1 H}-15 N NOE) as well as longitudinal (T 1 ) and transverse (T 2 ) relaxation times are affected by N-H bond dynamics and the molecule's rotational diffusion (58,59). Residues adopting regular secondary structure show heteronuclear NOE values close to the theoretical maximum (ϳ0.83 at a spectrometer frequency of 600 MHz), whereas values lower than ϳ0.65 are symptomatic of internal dynamics (43,60). The average heteronuclear NOE values for the PYD and CARD regions of ASC are high and similar (0.78 Ϯ 0.07 and 0.79 Ϯ 0.08, respectively) as expected for two rigid structures (Fig. 5A). In contrast, heteronuclear NOE values decrease from the PYD C terminus and the CARD N terminus toward the linker center (Fig. 5A). These results indicate that the linker undergoes local motions on a fast time scale compared with molecular tumbling. These motions become increasingly restricted toward the connections to the DDs. Thus, the heteronuclear NOE data indicate that ASC comprises two well ordered rigid domains connected by a flexible linker.  The next step is to analyze the dynamic behavior of each domain relative to the other. Two extreme models for ASC interdomain dynamics can be envisioned; in the first, both domains tumble as a single rigid body, and in the second model, each domain is dynamically independent. Backbone amide 15 N T 1 /T 2 ratios are particularly useful in this type of analysis, as they are similar among the domains when they tumble as a whole and different otherwise (43). The 15 N T 1 /T 2 ratios of the PYD and CARD of ASC are noticeably different, indicating that they reorient at different rates (Fig. 5B). In addition, NMR relaxation-derived c values of ASC individual domains are significantly larger than the predicted values (Table 1). Theoretical and NMR c values of globular proteins are generally in very good agreement. As an example, Table 1 shows the theoretical and experimental c values of the PYD-only protein ASC2 (56) of similar size and structure to ASC individual domains. The NMR c values of both PYD and CARD in ASC are also larger than the experimental c of ASC2, indicating that the former do not tumble independently. These results suggest that the PYD and CARD in ASC are in between the two extreme models, showing some interdomain flexibility and simultaneously sensing each domain the drag of the other.
This behavior is structurally illustrated by superimposing each individual domain of the NMR conformational ensemble (Fig. 6). The residual structural preferences of the linker result in a defined spatial interdomain organization that determines the orientation for binding of one domain relative to the other. Within this topological arrangement the flexibility of the linker increases the accessible space sampled by each domain, improving therefore, the chances to find interacting partners relative to proteins with two structurally fixed domains.
Interdomain motions caused by linker flexibility have been related to protein function. A classical example is the NMR study on the dynamic behavior of the two-domain protein calmodulin (43). This study shows that the long interdomain linker is highly mobile, supporting its role in calmodulin versatility to bind to multiple partners. Moreover, the linker flexibility is proposed to allow both protein halves to simultaneously interact with the target and to adopt the different domain orientations required in the formation of each complex. The binding requirements of ASC and calmodulin are analogous in that both proteins need to interact with different partners. Therefore, the flexibility of the linker could play a similar role in the interdomain dynamics of both proteins. Nevertheless, an important difference in the behavior of ASC could be that each domain binds at least one different target to form a particular complex. Like ASC, calmodulin domains show NMR relaxation-derived c values that are significantly larger than the prediction (Table 1) albeit smaller than the correlation time expected for a globular protein of similar size (43). Interdomain motions in calmodulin have been further studied, resulting in the determination of the motion time scale (61). Examples of proteins with domain dynamics fitting the first extreme scenario explained above have also been reported (62). In this case the domains orient together because short or rigid linkers connect them or because they participate in interdomain contacts. In the death domain superfamily, FADD serves as an example of two domains orienting as a whole (23).
A Model for ASC Oligomerization-ASC forms homo-and hetero-oligomeric assemblies that are tightly bound to its function. NMR data and AFM images reported here indicate that ASC is also capable of self-associating in vitro (Figs. 1 and 2). Based on this information and the propensity of death domains to form homotypic interactions within subfamilies, it is possible to build a model to illustrate how ASC could oligomerize. The model (Fig. 7A) uses the structure of full-length ASC as the monomer template together with the binding interface of Apaf-1-CARD-caspase-9-CARD complex structure (27), which is currently the single three-dimensional CARD/CARD complex structure known. The structure of a PYD/PYD complex has not been determined up to date.
The CARD/CARD interaction is asymmetric, involving helices 1 and 4 of one CARD and helices 2 and 3 of the other (Fig.  7A). The asymmetry leaves two free binding sites in the dimer: helices 1 and 4 of one monomer and helices 2 and 3 of the other. The free binding sites allow additional CARD/CARD interactions, naturally leading to oligomerization. PYD/PYD interactions are suggested to also involve helices 1 and 4 of one PYD and helices 2 and 3 of the other (25). The PYD/PYD interface would be asymmetric and, therefore, would leave free binding sites upon dimer formation. Thus, the PYDs could as well participate in the self-association of ASC through homophilic interactions. The formation of the CARD/CARD and PYD/ PYD interaction in the dimer model pre-establishes the relative binding orientation of the each domain to other partners (Fig.  7A). Moreover, in this model the pyrin domains are confined to a restrained space on top of the CARDs and do not obstruct the CARD/CARD interface (Fig. 7A). Interestingly, in the ASC structure the helices suggested to participate in the PYD/PYD interface are positioned as far as possible from the CARD and are, therefore, accessible for interactions with other partners (Fig. 7B). Thus, the CARD does not interfere with the PYD interface suggested to be involved in PYD/PYD interactions.
The CARDs central axes in the dimer model are positioned at an angle (Fig. 7A) that could result in the formation of a ring upon further association through the free interacting surfaces. The value of this angle (53.6°) is consistent with a ring composed of ϳ6 -7 monomers. Strikingly, electron microscopy data of the supramolecular apoptosome formed by Apaf-1 oligomerization show a 7-member CARD ring (63). In addition, the protein NALP1, which bears an N-terminal PYD and a C-terminal CARD flanking other domains, also forms a 7-fold symmetric ring as observed by electron microscopy (18). Both are rings of ϳ12-13-nm outer diameter. Interestingly, the disklike ASC oligomers (Fig. 2) of ϳ12-nm diameter suggest that ASC could oligomerize into rings analogous to those formed by Apaf-1 and NALP1. However, ASC has been shown by confocal microscopy to also form rather large specks of ϳ2 m in diameter (8) and filaments (2). These results together with the AFM data reported here suggest that the oligomerization of ASC could be a complex process. It is likely that the disk-like oligomers observed by AFM further aggregate into larger assemblies.

DISCUSSION
This study shows how the molecular architecture of ASC facilitates self-association and multiple binding to several proteins, which in turn could result in the assembly of supramolecular platforms. This role directly emerges from ASC interdomain orientation and dynamics. ASC interdomain topological organization facilitates binding by avoiding steric interference between the two domains and favors a specific protein binding orientation. In addition to spatial confinement, ASC shows interdomain flexibility, which is proposed to increase the search space of each domain independently, therefore, enhancing the probability to find interacting partners. The interdomain structural and dynamic properties of ASC are significantly different from FADD, which is the only other protein structure currently known with two death domains belonging to different subfamilies. The length of the linker (23 amino acids in ASC and 6 amino acids in FADD) could be partially responsible for the observed differences between the two proteins and, therefore, might emerge as an important factor in the operating mode of death domain proteins.
The oligomerization of ASC is another distinctive characteristic related to its capability to form supramolecular assemblies. The absence of intramolecular PYD/CARD interactions in the structure of ASC agrees with all structural and biochemical data of death domains reporting their tendency to form homophilic interactions within each subfamily and suggests that ASC oligomerizes through homotypic interactions mediated by its CARD and PYD. The overall dimension and shape of these oligomers are reported here. Interestingly, ASC oligomers are disk-like particles of similar size to the Apaf-1 CARD and NALP1 rings. Taken together, the structural and dynamic features of ASC shed light into the function of this protein as an adapter molecule and its capability to form supramolecular complexes in apoptosis and inflammation. Further research in this area will help to establish whether other members of the death domain superfamily, with multiple protein-protein binding domains connected by relatively long linkers, share some of these structural features and behave similarly.