Multiple Binding Sites on the Pyrin Domain of ASC Protein Allow Self-association and Interaction with NLRP3 Protein

A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.     

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Inflammasomes are multiprotein caspase‐activating complexes that enhance the maturation and release of proinflammatory cytokines (IL‐1β and IL‐18) in response to the invading pathogen and/or host‐derived cellular stress. These are assembled by the sensory proteins (viz NLRC4, NLRP1, NLRP3, and AIM‐2), adaptor protein (ASC), and effector molecule procaspase‐1. In NLRP3‐mediated inflammasome activation, ASC acts as a mediator between NLRP3 and procaspase‐1 for the transmission of signals. A series of homotypic protein‐protein interactions (NLRP3^PYD:ASC^PYD and ASC^CARD:CASP1^CARD) propagates the downstream signaling for the production of proinflammatory cytokines. Pyrin‐only protein 1 (POP1) is known to act as the regulator of inflammasome. It modulates the ASC‐mediated inflammasome assembly by interacting with pyrin domain (PYD) of ASC. However, despite similar electrostatic surface potential, the interaction of POP1 with NLRP3^PYD is obscured till date. Herein, to explore the possible PYD‐PYD interactions between NLRP3^PYD and POP1, a combined approach of protein‐protein docking and molecular dynamics simulation was adapted. The current study revealed that POP1's type‐Ia interface and type‐Ib interface of NLRP3^PYD might be crucial for 1:1 PYD‐PYD interaction. In addition to type‐I mode of interaction, we also observed type‐II and type‐III interaction modes in two different dynamically stable heterotrimeric complexes (POP1‐NLRP3‐NLRP3 and POP1‐NLRP3‐POP1). The inter‐residual/atomic distance calculation exposed several critical residues that possibly govern the said interaction, which need further investigation. Overall, the findings of this study will shed new light on hitherto concealed molecular mechanisms underlying NLRP3‐mediated inflammasome, which will have strong future therapeutic implications.     

Identification of Multifaceted Binding Modes for Pyrin and ASC Pyrin Domains Gives Insights into Pyrin Inflammasome Assembly

Inflammasomes are macromolecular complexes that mediate inflammatory and cell death responses to pathogens and cellular stress signals. Dysregulated inflammasome activation is associated with autoinflammatory syndromes and several common diseases. During inflammasome assembly, oligomerized cytosolic pattern recognition receptors recruit procaspase-1 and procaspase-8 via the adaptor protein ASC. Inflammasome assembly is mediated by pyrin domains (PYDs) and caspase recruitment domains, which are protein interaction domains of the death fold superfamily. However, the molecular details of their interactions are poorly understood. We have studied the interaction between ASC and pyrin PYDs that mediates ASC recruitment to the pyrin inflammasome, which is implicated in the pathogenesis of familial Mediterranean fever. We demonstrate that both the ASC and pyrin PYDs have multifaceted binding modes, involving three sites on pyrin PYD and two sites on ASC PYD. Molecular docking of pyrin-ASC PYD complexes showed that pyrin PYD can simultaneously interact with up to three ASC PYDs. Furthermore, ASC PYD can self-associate and interact with pyrin, consistent with previous reports that pyrin promotes ASC clustering to form a proinflammatory complex. Finally, the effects of familial Mediterranean fever-associated mutations, R42W and A89T, on structural and functional properties of pyrin PYD were investigated. The R42W mutation had a significant effect on structure and increased stability. Although the R42W mutant exhibited reduced interaction with ASC, it also bound less to the pyrin B-box domain responsible for autoinhibition and hence may be constitutively active. Our data give new insights into the binding modes of PYDs and inflammasome architecture.     

Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly

NALP3 inflammasome, composed of the three proteins NALP3, ASC, and Caspase-1, is a macromolecular complex responsible for the innate immune response against infection with bacterial and viral pathogens. Formation of the inflammasome can lead to the activation of inflammatory caspases, such as Caspase-1, which then activate pro-inflammatory cytokines by proteolytic cleavage. The assembly of the NALP3 inflammasome depends on the protein-interacting domain known as the death domain superfamily. NALP3 inflammasome is assembled via a pyrin domain (PYD)/PYD interaction between ASC and NALP3 and a caspase recruitment domain/caspase recruitment domain interaction between ASC and Caspase-1. As a first step toward elucidating the molecular mechanisms of inflammatory caspase activation by formation of inflammasome, we report the crystal structure of the PYD from NALP3 at 1.7-Å resolution. Although NALP3 PYD has the canonical six-helical bundle structural fold similar to other PYDs, the high resolution structure reveals the possible biologically important homodimeric interface and the dynamic properties of the fold. Comparison with other PYD structures shows both similarities and differences that may be functionally relevant. Structural and sequence analyses further implicate conserved surface residues in NALP3 PYD for ASC interaction and inflammasome assembly. The most interesting aspect of the structure was the unexpected disulfide bond between Cys-8 and Cys-108, which might be important for regulation of the activity of NALP3 by redox potential.     

Mapping of POP1-binding site on pyrin domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an essential adaptor protein in the formation of a multiprotein complex that activates procaspase-1. ASC is also known as a modulator of NF-kappaB activation pathways. ASC has a bipartite domain structure, consisting of an N-terminal pyrin domain (PYD) and a C-terminal caspase-recruitment domain. The PYD of ASC (ASC_PYD) is known to interact with various PYD-containing intracellular danger signal sensors and PYD-only proteins. Using purified proteins, we characterized the in vitro interaction of ASC_PYD with PYD-only protein 1 (POP1). POP1 specifically interacts with ASC_PYD with a dissociation constant of 4.08 +/- 0.52 microm but does not interact with Cryopyrin. NMR and mutagenesis experiments show that a negative electrostatic potential surface patch (EPSP) on ASC_PYD, consisting of the first (H1) and fourth (H4) helices, is essential in the interaction with POP1. A positive EPSP on POP1, consisting of the second (H2) and third (H3) helices, is a counterpart of this interaction. The interaction between ASC_PYD and POP1 is similar to the interaction between caspase recruitment domains of Apaf-1 and procaspase-9. In addition, we present evidence that conformational changes at the long loop of ASC_PYD between the H2 and H3 helices can affect its interaction with POP1. Based on our observations, we propose that the positive EPSP of ASC_PYD, including the H2 and H3 helices, may be the binding site for Cryopyrin, and the interaction with Cryopyrin may induce the dissociation of POP1 from ASC_PYD.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.     

Generalized semi-refolding methods for purification of the functional death domain superfamily

The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily and the pyrin domains (PYD) subfamily is one of the largest classes of protein interaction modules and plays a pivotal role in the apoptosis, inflammation, and immune cell signaling pathways. Despite the biological importance of the death domain superfamily, structural and in vitro biochemical studies have been limited because these domains are prone to aggregate under physiological conditions. Here, we describe a generalized method, termed semi-refolding, that is particularly applicable for purification of the functional death domain superfamily. The recombinant proteins Caspase-1 CARD, AIM2 PYD, NALP3 PYD, and RIP1 DD from inclusion bodies were successfully purified using this method.     

Three-Dimensional Structure of Human NLRP10/PYNOD Pyrin Domain Reveals a Homotypic Interaction Site Distinct from Its Mouse Homologue

NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a family of pattern-recognition receptors (PRRs) that sense intracellular microbial components and endogenous stress signals. NLRP10 (also known as PYNOD) is a unique NLRP member characterized by a lack of the putative ligand-binding leucine-rich repeat domain. Recently, human NLRP10 has been shown to inhibit the self-association of ASC into aggregates and ASC-mediated procaspase-1 processing. However, such activities are not found in mouse NLRP10. Here we report the solution structure and dynamics of human NLRP10 pyrin domain (PYD), whose helix H3 and loop H2–H3 adopt a conformation distinct from those of mouse NLRP10. Docking studies show that human and mouse NLRP10 PYDs may interact differently with ASC PYD. These results provide a possible structural explanation for the contrasting effect of NLRP10 on ASC aggregation in human cells versus mouse models. Finally, we also provide evidence that in human NLRP10 the PYD domain may not interact with the NOD domain to regulate its intrinsic nucleotide hydrolysis activity.     

Crystal Structure of the F27G AIM2 PYD Mutant and Similarities of Its Self-Association to DED/DED Interactions

Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome. The activated caspases process proinflammatory cytokines IL-1β and IL-18 and induce the inflammatory form of cell death pyroptosis. Here we show that AIM PYD (AIM2^PYD) self-oligomerizes. We notice significant sequence homology of AIM2^PYD with the hydrophobic patches of death effector domain (DED)-containing proteins and confirm that mutations on these residues disrupt AIM2^PYD self-association. The crystal structure at 1.82 Å resolution of such a mutant, F27G of AIM2^PYD, shows the canonical six-helix (H1–H6) bundle fold in the death domain superfamily. In contrast to the wild-type AIM2^PYD structure crystallized in fusion with the large maltose-binding protein tag, the H2–H3 region of the AIM2^PYD F27G is well defined with low B-factors. Structural analysis shows that the conserved hydrophobic patches engage in a type I interaction that has been observed in DED/DED and other death domain superfamily interactions. While previous mutagenesis studies of PYDs point to the involvement of charged interactions, our results reveal the importance of hydrophobic interactions in the same interfaces. These centrally localized hydrophobic residues within fairly charged patches may form the hot spots in AIM2^PYD self-association and may represent a common mode of PYD/PYD interactions in general.     

ASC,which is composed of a PYD and a CARD,is up-regulated by inflammation and apoptosis in human neutrophils

ASC is an adaptor protein that is composed of two protein-protein interaction domains, a PYRIN domain (PYD), and a caspase-recruitment domain (CARD). Recently, ASC was identified as a binding partner of pyrin, which is the product of MEFV, a gene causing familial Mediterranean fever (FMF). Mutations in MEFV result in defects in control of neutrophil-mediated inflammation. Thus we focused on the expression of ASC in neutrophils. Immunohistochemical study showed that ASC is increased in neutrophils in severe inflammatory sites of gangrenous appendicitis. We, then, tested whether proinflammatory mediators induce ASC using peripheral blood neutrophils in vitro. ASC expression was transiently up-regulated by IL-1alpha, IL-1beta, IFN-alpha, IFN-gamma, TNFalpha, and LPS. ASC was also increased by incubation with either anti-Fas antibody or recombinant soluble Fas ligand. The Fas-mediated induction of ASC was inhibited by a general caspase inhibitor, z-VAD-fmk, and an immunocytochemical study showed that ASC was increased in neutrophils exhibiting characteristic phenotypes for apoptosis. These findings suggest that up-regulation of ASC is closely associated with inflammation and apoptosis in neutrophils.     

Inflammasome regulation by adaptor isoforms,ASC and ASCb,via differential self-assembly

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular docking with the procaspase-1 CARD domain. Altogether, our results indicate that ASC’s faster and less polydisperse polymerization is more efficient, plausibly explaining inflammasome activation differences by ASC isoforms at the molecular level.     

The pyrin inflammasome in host–microbe interactions

A) RhoA regulation of pyrin. B) Inactivation of RhoA and control of pyrin inflammasome activation by bacterial effectors and toxins.A) In steady-state conditions, RhoA is constantly being turned on and off by GEFs and GAPs that exchange GDP for GTP or accelerate conversion of GTP to GDP, respectively. RhoA regulates the pyrin inflammasome through interactions with transducers of RhoA signaling, PRKs, that phosphorylate pyrin creating a 14-3-3 binding site. Phosphorylated and 14-3-3 bound pyrin is locked in an off state where its conformation prevents interaction with inflammasome components. B) (1) Bacteria deliver toxins or effecter proteins to host cells using T3SS, T6SS, secretion via ECVs or other general secretion mechanisms. There are several classes of RhoA modifying toxins including, those that covalently modify RhoA, those that proteolytically cleave RhoA and GAPs. (2) Toxins and effectors inactivate RhoA stopping PRK signaling to pyrin. (3) The lack of negative signal by PRKs and possible action of a phosphatase leads to the dephosphorylation of pyrin on its regulatory serine residues and loss of 14-3-3 binding. (4) Another class of toxins and effectors are those that inhibit pyrin inflammasome formation. YopM, an inhibitor of pyrin inflammasome formation represented in blue, recruits kinases RSK and PRK to pyrin to keep it phosphorylated. (5) When pyrin is dephosphorylated it then binds to the ASC adapter protein which binds and dimerizes pro-caspase-1 forming the inflammasome complex. Additionally, evidence suggests that microtubule polymerization is required for inflammasome formation. Upon dimerization, pro-caspase-1 undergoes autoproteolytic cleavage fully activating the enzyme. Mature caspase-1 then cleaves pro-IL-1β, pro-IL-18 and GSDMD to their mature forms. (6) Gasdermin-D then oligomerizes and forms pores in the plasma membrane which allows secretion of IL-1β and IL-18. If enough pores in the plasma membrane form the cell will undergo cell death via lysis known as pyroptosis.Abbreviations: GDP: guanosine diphosphate; GTP: guanosine triphosphate; PRK: protein kinase C-related kinase; T3SS: type 3 secretion system; T6SS: type 6 secretion system; ECVs: extracellular vesicles; GAP: GTPase activating proteins; ASC: apoptosis-associated speck-like protein; IL: interleukin; GSDMD: gasdermin-D; PSP: protein serine/threonine phosphatase.

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AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks'' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.     

凋亡相关斑点样蛋白的研究进展

凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD,ASC）是一种含有N端热蛋白样结构域和C端胱天氨酸募集结构域的接头分子。ASC可以通过它含有的同源蛋白互作结构域PYD和CARD的寡聚化来募集上下游与其含有同源结构域的其他蛋白,从而参与多条信号转导途径,在炎症反应、肿瘤发生、细胞凋亡和NF-κB信号通路的调节方面发挥重要的生物学作用。     

Single-Molecule Fluorescence Reveals the Oligomerization and Folding Steps Driving the Prion-like Behavior of ASC

Single-molecule fluorescence has the unique ability to quantify small oligomers and track conformational changes at a single-protein level. Here we tackled one of the most extreme protein behaviors, found recently in an inflammation pathway. Upon danger recognition in the cytosol, NLRP3 recruits its signaling adaptor, ASC. ASC start polymerizing in a prion-like manner and the system goes in “overdrive” by producing a single micron-sized “speck.” By precisely controlling protein expression levels in an in vitro translation system, we could trigger the polymerization of ASC and mimic formation of specks in the absence of inflammasome nucleators. We utilized single-molecule spectroscopy to fully characterize prion-like behaviors and self-propagation of ASC fibrils. We next used our controlled system to monitor the conformational changes of ASC upon fibrillation. Indeed, ASC consists of a PYD and CARD domains, separated by a flexible linker. Individually, both domains have been found to form fibrils, but the structure of the polymers formed by the full-length ASC proteins remains elusive. For the first time, using single-molecule Förster resonance energy transfer, we studied the relative positions of the CARD and PYD domains of full-length ASC. An unexpectedly large conformational change occurred upon ASC fibrillation, suggesting that the CARD domain folds back onto the PYD domain. However, contradicting current models, the “prion-like” conformer was not initiated by binding of ASC to the NLRP3 platform. Rather, using a new method, hybrid between Photon Counting Histogram and Number and Brightness analysis, we showed that NLRP3 forms hexamers with self-binding affinities around 300 nM. Overall our data suggest a new mechanism, where NLRP3 can initiate ASC polymerization simply by increasing the local concentration of ASC above a supercritical level.     

NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.     

1H, 13C and 15N resonance assignments of the pyrin domain from human PYNOD

PYNOD is a novel protein belonging to a large family of proteins containing the nucleotide-binding and oligomerization domain (NOD) involved in inflammation and apoptosis. Human PYNOD inhibits inflammatory response mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). Here we report the ¹H, ¹³C and ¹⁵N resonance assignments and secondary structure identification of the pyrin domain (PYD) of human PYNOD as the first step towards elucidating the structural basis of the anti-inflammatory activity of PYNOD.