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.     

Fire and death: the pyrin domain joins the death-domain superfamily

Apoptosis and inflammation are important cellular processes that are highly regulated through specific protein-protein interactions (PPI). Proteins involved in these signaling cascades often carry PPI domains that belong to the death-domain superfamily. This includes the structurally well-characterized Death Domain (DD), the Death Effector Domain (DED) and the Caspase Recruitment Domain (CARD) subfamilies. Recently, a fourth member of the DD superfamily was identified, the Pyrin Domain (PYD). Based on sequence alignments, homology to other domains occurring in death-signalling pathways, and secondary-structure prediction, the PYD was predicted to have an overall fold similar to other DD superfamily members. Just recently, NMR structures of two PYDs have been determined. The PYD structures not only revealed the DD superfamily fold as previously predicted, but also distinct features that are characteristic exclusively for this subfamily. This review summarizes recent findings and developments regarding structural aspects of the DD superfamily, with a special emphasis on the PPIs of the DD superfamily.     

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.     

Thermodynamics and stability of the PAAD/DAPIN/PYRIN domain of IFI-16

The PAAD domain is a conserved domain recently identified in more than 35 human proteins that are involved in apoptosis and inflammatory signaling pathways. Structural studies have confirmed that this domain belongs to the death domain superfamily which includes PAAD/CARD/DED/DD families. Recently, the 3D structures determined by NMR of NALP1 and ASC PAAD domain, members of the PAAD family, have shown that it is composed of a 6 helix bundle as with other death domain family members. However, helix-3 in the solved structures is unordered in solution. In this study we compare the thermodynamic, folding and stability properties of different members of the PAAD and CARD families and investigate structural conformational changes induced by the helix inducers trifluoroethanol and SDS on the PAAD domain of IFI16 and on the CARD domain of RAIDD. We show that inside the PAAD and CARD families, members have similar thermodynamic properties, however, the DeltaG of folding for PAAD and CARD members are, respectively, -1.4 and -5.5 kcal mol(-1). This difference is attributed to less alpha helical content for PAAD due to the unfolding of helix-3 that lowers bonded energy and increases disorder when compared to CARD members. Despite identical fold between PAAD and CARD families but limited sequence identity, there are striking differences in the thermodynamics of both families.     

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.     

The death domain superfamily: a tale of two interfaces?

The death domain superfamily, composed of the death domain (DD), death effector domain (DED) and caspase recruitment domain (CARD) families of proteins, plays a pivotal role in signaling events that regulate apoptosis. This review compares and contrasts the ten superfamily members with known structures. In particular, the two heterodimerization modes described to date, the CARD-CARD interaction between human Apaf-1 and procaspase 9, and the DD-DD interaction between Drosophila Pelle and Tube, are examined. The dimerization modes are strikingly different and, importantly, are not mutually exclusive. In fact, a trimer can be formed using both interactions.     

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.     

Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition

The death-inducing signaling complex (DISC) comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here, we report the crystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. It reveals a noncanonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a different mode of interaction in the DD superfamily. Whereas the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher-order oligomerization required for caspase activation in the DISC.     

The PYRIN domain: a member of the death domain-fold superfamily

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.     

Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions

Death-fold domains constitute an evolutionarily conserved superfamily that mediates apoptotic signaling. These motifs, including CARD (caspase recruitment domain), DD (death domain), and DED (death effector domain), are believed to exert their effects solely through homotypic interactions. Herein we demonstrate that the CARD-containing protein ARC engages in nontraditional death-fold interactions to suppress both extrinsic and intrinsic death pathways. The extrinsic pathway is disrupted by heterotypic interactions between ARC's CARD and the DDs of Fas and FADD, which inhibit Fas-FADD binding and assembly of the death-inducing signaling complex (DISC). The intrinsic pathway is antagonized by ARC-Bax binding, involving ARC's CARD and the Bax C terminus. This inhibits Bax activation and translocation to the mitochondria. Knockdown of endogenous ARC facilitates DISC assembly and triggers spontaneous Bax activation and apoptosis. Conversely, physiological levels of ARC suppress these events. These studies establish a critical role for nonhomotypic death-fold interactions in the regulation of apoptosis.     

P45 Forms a Complex with FADD and Promotes Neuronal Cell Survival Following Spinal Cord Injury

Fas-associated death domain (DD) adaptor (FADD), a member of the DD superfamily, contains both a DD and a death effector domain (DED) that are important in mediating FAS ligand-induced apoptotic signaling. P45 is a unique member of the DD superfamily in that it has a domain with sequence and structural characteristics of both DD and DED. We show that p45 forms a complex with FADD and diminishes Fas-FADD mediated death signaling. The DED of FADD is required for the complex formation with p45. Following spinal cord injury, transgenic mice over-expressing p45 exhibit increased neuronal survival, decreased retraction of corticospinal tract fibers and improved functional recovery. Understanding p45-mediated cellular and molecular mechanisms may provide insights into facilitating nerve regeneration in humans.     

Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase‐1

The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N‐terminal pyrin domain (PYD) and a C‐terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase‐1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose‐binding protein. The structure reveals the six‐helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase‐1 CARD model suggests potential mechanisms for their association through electrostatic attraction. Proteins 2013; 81:1266–1270. © 2013 Wiley Periodicals, Inc.     

Solution structure of the tumor necrosis factor receptor-1 death domain.

Tumor necrosis factor receptor-1 death domain (TNFR-1 DD) is the intracellular functional domain responsible for the receptor signaling activities. The solution structure of the R347K mutant of TNFR-1 DD was solved by NMR spectroscopy. A total of 20 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 1167 distance constraints and 117 torsion angle constraints. The atomic rms distribution about the mean coordinate positions for the 20 structures for residues composing the secondary structure region is 0.40 A for the backbone atoms and 1.09 A for all atoms. The structure consists of six antiparallel alpha-helices arranged in a similar fashion to the other members of the death domain superfamily. The secondary structure and three-dimensional structure of R347K TNFR1-DD are very similar to the secondary structure and deduced topology of the R347A TNFR1-DD mutant. Mutagenesis studies identified critical residues located in alpha2 and part of alpha3 and alpha4 that are crucial for self-interaction and interaction with TRADD. Structural superposition with previously solved proteins in the death domain superfamily reveals that the major differences between the structures reside in alpha2, alpha3, and alpha4. Interestingly, these regions correspond to the binding sites of TNFR1-DD, providing a structural basis for the specificity of death domain interactions and its subsequent signaling event.     

Crystal structure of RAIDD death domain implicates potential mechanism of PIDDosome assembly

Caspase-2 is implicated in stress-induced apoptosis that acts as an upstream initiator of mitochondrial permeabilization. Recent studies have shown that caspase-2 activation requires a molecular complex known as the PIDDosome comprising the p53-inducible protein PIDD, the adapter protein RAIDD and caspase-2. RAIDD has an N-terminal caspase recruitment domain (CARD) that interacts with the CARD of caspase-2 and a C-terminal death domain (DD) that interacts with the DD in PIDD. As a first step towards elucidating the molecular mechanisms of caspase-2 activation, we report the crystal structure of RAIDD DD at 2.0 A resolution. The high-resolution structure reveals important features of RAIDD DD that may be important for DD folding and dynamics and for assembly of the PIDDosome.     

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.     

Homology modeling provides insights into the binding mode of the PAAD/DAPIN/pyrin domain,a fourth member of the CARD/DD/DED domain family

The PAAD/DAPIN/pyrin domain is the fourth member of the death domain superfamily, but unlike other members of this family, it is involved not only in apoptosis but also in innate immunity and several other processes. We have identified 40 PAAD domain-containing proteins by extensively searching the genomes of higher eukaryotes and viruses. Phylogenetic analyses suggest that there are five categories of PAAD domains that correlate with the domain architecture of the entire proteins. Homology models built on CARD and DD structures identified functionally important residues by studying conservation patterns on the surface of the models. Surface maps of each subfamily show different distributions of these residues, suggesting that domains from different subfamilies do not interact with each other, forming independent regulatory networks. Helix3 of PAAD is predicted to be critical for dimerization. Multiple alignment analysis and modeling suggest that it may be partly disordered, following a new paradigm for interaction proteins that are stabilized by protein-protein interactions.     

In vitro reconstitution of the interactions in the PIDDosome

Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.     

Determining the 3D structure of human ASC2 protein involved in apoptosis and inflammation

ASC2 structure has been well defined by 1141 NOE experimental restraints. The model consists of five alpha helices. alpha-Helices are connected by short random structure loops. The sole exception is the loop connecting helices 2 and 3, which has a 20-residue length. Folding generally agrees with the folding of recently published death domain structures in which alpha-helix structures have been reported. In spite of structural similarity, amino acid sequence homology with the most similar protein (ASC1) is just 64%. DD, DED, and CASP protein structures present six helices along their sequences; ASC2 presents 5 well-defined helices due to long distance restraints. However, a helical fragment was observed between amino acids 38 and 42 (representing helix 3) in the death domains when constructing the model.     

Recognition of ERK MAP kinase by PEA-15 reveals a common docking site within the death domain and death effector domain

PEA-15 is a multifunctional protein that modulates signaling pathways which control cell proliferation and cell death. In particular, PEA-15 regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been determined using NMR spectroscopy and its interaction with ERK defined by characterization of mutants that modulate ERK function. PEA-15 is composed of an N-terminal death effector domain (DED) and a C-terminal tail of irregular structure. NMR 'footprinting' and mutagenesis identified elements of both the DED and tail that are required for ERK binding. Comparison of the DED-binding surface for ERK2 with the death domain (DD)-binding surface of Drosophila Tube revealed an unexpected similarity between the interaction modes of the DD and DED motifs in these proteins. Despite a lack of functional or sequence similarity between PEA-15 and Tube, these proteins utilize a common surface of the structurally similar DD and DED to recognize functionally diverse targets.