Death, TIR, and RHIM: Self‐assembling domains involved in innate immunity and cell‐death signaling

The innate immune system consists of pattern recognition receptors (PRRs) that detect pathogen‐ and endogenous danger‐associated molecular patterns (PAMPs and DAMPs), initiating signaling pathways that lead to the induction of cytokine expression, processing of pro‐inflammatory cytokines, and induction of cell‐death responses. An emerging concept in these pathways and associated processes is signaling by cooperative assembly formation (SCAF), which involves formation of higher order oligomeric complexes, and enables rapid and strongly amplified signaling responses to minute amounts of stimulus. Many of these signalosomes assemble through homotypic interactions of members of the death‐fold (DF) superfamily, Toll/IL‐1 receptor (TIR) domains, or the RIP homotypic interaction motifs (RHIM). We review the current understanding of the structure and function of these domains and their molecular interactions with a particular focus on higher order assemblies.


DEATH-FOLD DOMAIN SUPERFAMILY
The DF superfamily is composed of 4 subfamilies, the 'death domain' (DD), the 'death effector domain' (DED), the 'caspase recruitment domain' (CARD), and the 'pyrin domain' (PYD), which are all known to form homotypic interactions leading to the formation of oligomeric signaling assemblies such as inflammasomes and the Myddosome (Fig. 2).
These domains are found in many multicellular organisms including mammals, Drosophila, zebrafish and C. elegans. DED, CARD, and PYD containing proteins are also found in some viral pathogens, where they are involved in host defense system evasion (reviewed in [28]).
Members of the DF superfamily share a common -helix bundle (usually 6 helices, H1-H6), with helices arranged anti-parallel in a Greek key type topology surrounding a hydrophobic core (Fig. 3).
The arrangement of these helices results in 6 protein binding surfaces, which mediate cooperative interactions with 3 types of interfaces (type I, II, and III). These interfaces facilitate the cooperative assembly of large, oligomeric signaling complexes, and the recruitment of effector enzymes, such as caspases and kinases, which undergo concentration-dependent proximity-induced auto-activation following assembly. Proteins rarely contain the DF domain in isolation, typically this domain is found in combination with other protein-protein interaction domains or effector domains conferring enzymatic activity.
Although all members of the DF superfamily share the same overall fold, the individual subfamilies exhibit distinct structural and sequence differences that confer a degree of specificity. Members of each subfamily interact with a specific array of binding partners. Typically, these are homotypic interactions such as that of 'apoptosis-associated speck-like protein containing a caspase recruitment domain' (ASC) CARD recruiting procaspase-1 CARD, and the DD of the TLR adaptor protein 'myeloid differentiation primary response gene 88' (MyD88) interacting with the DDs of the 'IL-1R associated kinase' (IRAK) -2 and -4. However, the CARD of 'apoptosis repressor with a CARD protein' (ARC) has been reported to interact with the DDs of the 'Fas-associating death domain-containing protein' (FADD) in vitro, 29 and the recruitment of procaspase-8 to inflammasomes via interaction between the ASC PYD and the procaspase tandem DED suggests heterotypic interactions also play a role in the formation of signaling complexes. 30

The DD subfamily
The DD serves as a homotypic protein-protein interaction domain in numerous intracellular signaling proteins involved in innate immunity and cell-death pathways (Fig. 1). Members of the death receptor subfamily of the 'tumor necrosis factor receptor' (TNFR) superfamily mediate cell-death pathways following ligand-induced oligomerization of extracellular receptor domains and subsequent recruitment of DD containing proteins through their own intracellular DDs. The death receptors, 'first apoptotic signal receptor' (Fas), 'death receptor 4' (DR4), and 'death receptor 5' (DR5) interact via a homotypic interaction with the C-terminal DD of FADD. FADD also contains an N-terminal DED and homotypically interacts with the 'tandem DEDs' (tDEDs) of procaspase-8 or -10 to form the 'death signaling complex' (DISC). Assembly of the complex activates the initiator caspases that in turn activate effector caspases, such as caspase-3 and -7, leading to apoptosis. [31][32][33][34][35] Two other death receptors, TNFR1 and 'death receptor 3' (DR3), recruit the adaptor protein 'TNF receptor type 1-associated protein with death domain' (TRADD) via the N-terminal DD of TRADD.
Recruitment of TRADD can facilitate either pro-cell survival and 'nuclear factor kappa-light-chain-enhancer of activated B cells' (NF-B) activation through the DD:DD recruitment of RIP1 and 'TNFR associated factor 2' (TRAF2) termed complex I, or when RIP1 is not ubiquitylated, initiation of apoptosis via DD:DD mediated recruitment of FADD and subsequently procaspase-8, termed complex II. [36][37][38][39] Outside of the TNFR superfamily, the DD-containing proteins, 'p53-induced protein with a DD' (PIDD) and 'RIP-associated ICH-1 homologous protein with a death domain' (RAIDD), form an assembly termed the PIDDosome via homotypic interactions between the DDs of PIDD and RAIDD, and between the N-terminal CARDs of RAIDD and procaspase-2. Similar to TRADD signaling, isoforms of PIDD have been reported to recruit RIP1, leading to NF-B activation and cell survival. 40,41 In addition to a C-terminal TIR domain, the TLR adaptor protein MyD88 possesses an N-terminal DD, which facilitates the assembly of an oligomeric assembly complex, termed the Myddosome, through DD:DD interactions with IRAKs, which contain an N-terminal DD and a carboxy-terminal Ser/Thr kinase or pseudokinase domain (Fig. 2).
The 'MyD88 adaptor-like protein' (MAL) TIR domain nucleates the formation of MyD88 assemblies, 5 suggesting that oligomerization of the MyD88 TIR domain serves to cluster the MyD88 DDs, followed by recruitment and proximity based auto-and cross-phosphorylation appears to have no catalytic function, acting as a modulator through protein-protein interactions. In particular, PEA-15 appears to inhibit apoptosis by binding to FADD through homotypic DD interactions and preventing the recruitment and activation of procaspases. [10][11][12]24 However, PEA-15 is also implicated in 'mitogen-activated protein kinase' (MAPK) signaling through a non-DD interaction with 'extracellular signal-regulated kinase' (ERK). DEDD and DEDD2 translocate to the cell nucleus and induce limited apoptotic signaling; however, when nuclear localization is prevented, DEDD and DEDD2 bind to procaspase-8 or -10 in a DED-dependent manner and induce apoptosis. The significance of this signaling in vivo remains unknown. 46 The structures of the DEDs of FADD, 47  other DF domains, DEDs contain a surface-exposed hydrophobic patch formed predominantly by residues on helix H2, and a E/D-RxDLmotif, with the N-terminal acidic residue contributed by helix H2, and the C-terminal motif by helix H6. Also, tDEDs have an additional helix H7 at the C-terminus of DED1, linking DED1 to the second DED (DED2). 47,[50][51][52] The DEDs of FADD and cFLIP, and tDEDs of procaspase-8 have been shown to form filamentous assemblies in vitro. 49 Comparison of the cryo-EM structure of procaspase-8 tDED assembly with the crystal structure suggests that the tDEDs undergo little to no conformational changes upon oligomerization. 49,50 FADD DED filaments may act as a template for helical association of procaspase-8 tDED in a mechanism similar to that reported for RIG-I:

The CARD subfamily
The CARD subfamily ( Fig. 1) was first identified in the apoptotic signaling proteins RAIDD, caspase-2, and 'cell death protein 3' (CED-3), an orthologue of caspase-9, due to sequence similarity and their known involvement in apoptotic signaling. 53 Originally described as a motif that facilitated the recruitment of caspases to an apoptotic signaling complex, CARD-containing proteins are now known to have a broad range of functions in multiple immune system signaling pathways. The roles of CARDs can be broadly classified into 3 groups: the CARDs found as pro-domains of caspases, CARDs that act as receptors or adaptors in the assembly of signaling complexes, and CARDs that inhibit or otherwise modulate signaling complexes.
The CARD-containing proteins 'apoptotic protease activating factor-1' (Apaf-1), 54 RIG-I, 9,55  The monomeric structures of the PYDs of AIM2, 69 ASC, 71 pyrin, 74 and various NLRPs have been determined by either nuclear magnetic resonance (NMR) or crystallography. In addition to the characteristic DF, PYDs display a short 3 helix and an elongated 2-3 loop compared to other DF subfamilies. In the case of the ASC PYD, the acidic conditions used for NMR spectroscopy appear to have abolished self-association, and thus provide little insight into the interfaces of the PYD assemblies. However, the cryo-EM structure of the ASC PYD filament at near-atomic resolution demonstrates that few conformational changes are apparent between monomeric PYD and those of the filament. In the filament, the 3 helix and 2-3 loops appear to be stabilized by the type I, II, and III interfaces. The ASC PYD filaments display a 3-fold helical symmetry, when looking down the helical axis, featuring a hollow center with an inner diameter of ∼ 20Å and outer diameter of ∼90Å. 12 The crystal structure of the AIM2 PYD displays little difference to that of the ASC and NLRP3 PYDs, with the exception of the 1 and 6 helices, which are elongated in the AIM2 PYD. 12,69,72 PYDs typically display highly positively or negatively charged surface regions, which are either involved in self-association or conferring specificity for a particular binding partner. 12,69

Death-fold family oligomerization interfaces
The DF domains display 3 shared asymmetric non-overlapping interfaces that can potentially interact with up to 6 different binding partners (Fig. 3). The interfaces between layers or strands of the DF assemblies may differ depending on the symmetry or oligomerization pattern used to describe the assembly, for instance the Myddosome 42 and procaspase-1 CARD assemblies 65

TIR DOMAINS
The TIR domain is a small globular domain consisting of  residues, and in animals and plants it is usually found in multidomain proteins involved in innate immunity pathways (Fig. 1). Many bacterial proteins also contain TIR domains, and at least some of them are associated with virulence by suppressing host innate immunity signaling pathways.
In mammals TIR domains are found (i) on the cytosolic side of TLRs, human 'IL-1R accessory protein-like 1' (IL-1RAPL) 100 ; the Toll-related receptor TRR-2 from the lower metazoan Hydra magnipapillata (PDB  85,96,97 In addition, SEFIR domain structures are available for the human IL-17RA and IL-17RB receptors 106,107 and the Bacillus cereus protein BcSEFIR. 108 The structural core of all TIR domains are conserved but there are significant structural differences in the surrounding loops and -helices (size, number, and orientation), in particular between the different sub-types of TIR domains (TLRs, TLR-adaptors, IL-1Rs, plant, and bacteria) (reviewed in [109]). For example, in plant TIR domains the region between the D and E strands contains 3 well-defined helices ( D1, D2, and D3) and is significantly different compared to both mammalian and bacterial TIR domains, which only contain 1 or 2 short helices (Fig. 4A). The central 5-stranded sheet of SEFIR domains as well as the 3 helices A, B, and E superimpose well with the TLR receptor TIR domains, but there are significant structural differences in the CC region, the C and D helices, and the DD region (Fig. 4A). involves residues in the DD and EE loops. 85,96,97,105 The BB loops from the 2 interacting molecules are exposed to the solvent for possible interaction with host molecules.

TIR domain enzyme activity
SARM has been shown to have TLR-independent roles in neurons, 79,80 and the local cell death program induced by SARM after axonal injury involves rapid breakdown of 'nicotinamide adenine dinucleotide' (NAD + ) into nicotinamide and ADP-ribose. 111   activity. Interestingly, the Act1 SEFIR domain has recently been shown to directly bind and stabilize mRNAs encoding key inflammatory proteins, 114 suggesting that nucleotide binding may be a common feature among TIR and SEFIR domains.

RHIM REGIONS
The 'RIP homotypic interaction motif' (RHIM) is a short sequence of ∼15-20 amino acids, first identified as a homotypic interaction motif that facilitates association between RIP1 and RIP3. 115 Located at the C-terminus of RIP3 and adjacent to the C-terminal DD in RIP1, the RHIM domain forms the core of the amyloid signaling complex known as the necrosome, which coordinates necroptosis, a regulated form of necrosis that occurs upon inhibition of caspase-8 mediated apoptosis. 16,[115][116][117] The RHIM region contains a core motif of (V/I)-Q-(V/I/L/C)-G, and is also found in 'DNA-dependent activator of IFN regulatory factors' (DAI), 118 16,18,116,117,[124][125][126][127] . In addition to RIP1-mediated necroptosis, TRIF and DAI have been reported to recruit RIP3, resulting in MLKL activation and necroptosis independent of RIP1, 118-120 while ICP6 and ICP10 appear to interact with RIP1 and RIP3 and disrupt necroptosis in a RHIM-dependent manner. 122 Structures of the RHIM regions are limited to the crystal structure of a homo-amyloid formed by the RIP3 tetrad VQVG and the solid-state NMR structure of the RIP1/RIP3 hetero-amyloid complex (Fig. 4C). 18 Both the RIP3 homo-amyloid and RIP1/RIP3 heteroamyloid fibril feature two parallel -sheets arranged together in an anti-parallel fashion, with the hydrophobic residues of the (V/I)-Q-(V/I/L/C)-G tetrad forming a hydrophobic core. The overall structure of the RIP1:RIP3 hetero-amyloid complex further reveals that each strand in the parallel -sheet is broken into 4 short -strand segments separated by short turns. In the RIP1:RIP3 hetero-amyloid fibril, the parallel strands of the -sheets are formed from alternating RIP1:RIP3 RHIM domains. In addition to the hydrophobic core, stacking of the parallel RIP1:RIP3 RHIM domains is supported by Asn and Gln ladders formed between the side chains of Asn535 in RIP1 and Asn454 in RIP3 and the side chains of RIP1 residue Gln540 and RIP3 residue Gln459. Additional interactions including tyrosine stacking interactions formed between Tyr534 (RIP1) and Tyr453 (RIP3), and a Ser536 (RIP1)/Cys455 (RIP3) ladder further stabilize the assembly. 18

CONCLUDING REMARKS
In innate immunity and cell-death pathways, SCAF is emerging as a key signaling mechanism. The first characterized examples of SCAF involved members of the DF superfamily, but more recently RHIM regions and TIR domains have also been shown to form open-ended assemblies and to engage in SCAF. The higher order assemblies formed by these domains have distinct properties. The RHIM assemblies resemble amyloids and prions, in which adjacent interacting elements are tightly packed, resulting in cooperative contacts and a high barrier of dissociation. The DF signalosomes consist of folded domains, but cooperative interactions among 6 weakly associating adjacent surfaces lead to assemblies that can be as stable as amyloids. 8,12 By contrast, the 2-stranded head-to-tail assemblies of TIR domains have lower valency and are less stable. The MAL filament, for example, disassembles at low temperatures in solution. 3 The static and dynamic properties associated with the different assemblies could be directly related to the function of the assemblies and the biological outcome of the signaling pathways they are involved in. For example, in TLR signaling, it is likely that the low valency and observed dynamic properties of TIR domain assemblies are important for the observed rapid assembly and disassembly of TLR4:MAL:MyD88 complexes in macrophages. 110 On the other hand, DF and RHIM assemblies may persist even after cell lysis.

DISCLOSURE
The authors declare no conflicts of interest.