A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing.

Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma.

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1.

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel ß-sheets of seven and six strands, arranged as a distorted ß-sandwich. Furthermore, two long loops partially cover the concave face of the ß-sandwich defined by the ß-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome.

Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicansADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences.IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicansADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

Structural basis for the regulation of inducible nitric oxide synthase by the SPRY domain-containing SOCS box protein SPSB2, an E3 ubiquitin ligase.

Relatively high concentration of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in response to a variety of stimuli is a source of reactive nitrogen species, an important weapon of host innate immune defense. The SPRY domain-containing SOCS box protein 2 (SPSB2) is an E3 ubiquitin ligase that regulates the lifetime of iNOS. SPSB2 interacts with the N-terminal region of iNOS via a binding site on the SPRY domain of SPSB2, and recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, leading to its proteasomal degradation. Although critical residues for the SPSB2-iNOS interaction have been identified, structural basis for the interaction remains to be explicitly determined. In this study, we have determined a crystal structure of the N-terminal region of iNOS in complex with the SPRY domain of SPSB2 at 1.24 Å resolution. We have resolved the roles of some flanking residues, whose contribution to the SPSB2-iNOS interaction was structurally unclear previously. Furthermore, we have evaluated the effects of SPSB2 inhibitors on NO production using transient transfection and cell-penetrating peptide approaches, and found that such inhibitors can elevate NO production in RAW264.7 macrophages. These results thus provide a useful basis for the development of potent SPSB2 inhibitors as well as recruiting ligands for proteolysis targeting chimera (PROTAC) design.

PAM-less plant genome editing using a CRISPR-SpRY toolbox.

The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research1-6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.

Substrate recognition by TRIM and TRIM-like proteins in innate immunity.

TRIM (Tripartite motif) and TRIM-like proteins have emerged as an important class of E3 ligases in innate immunity. Their functions range from activation or regulation of innate immune signaling pathway to direct detection and restriction of pathogens. Despite the importance, molecular mechanisms for many TRIM/TRIM-like proteins remain poorly characterized, in part due to challenges of identifying their substrates. In this review, we discuss several TRIM/TRIM-like proteins in RNA sensing pathways and viral restriction functions. We focus on those containing PRY-SPRY, the domain most frequently used for substrate recognition, and discuss emerging mechanisms that are commonly utilized by several TRIM/TRIM-like proteins to tightly control their interaction with the substrates.

Crystal structure of the SPRY domain-containing protein 7 reveals unique structural features.

The SPRY/B30.2 domain is one of the most abundant protein domains found in eukaryotes. Vast majority of the SPRY domain-containing proteins are multi-domain proteins. The SPRY domain-containing protein 7 (SPRY7, also named C13orf1, and named chronic lymphocytic leukemia deletion region gene 6 protein, CCLD6, encoded by the spryd7 gene) is the smallest SPRY domain protein in human that does not contain other accessory domains. Here we have determined the crystal structure of human SPRY7 at a resolution of 1.62 Å and found that SPRY7 has some unique structural features that are not present in other previously reported SRPY domain structures. Overall, SPRY7 may represent an evolutionary early version of the SPRY domain, and subsequent loop insertions and expansions, residue substitutions, as well as domain combinations have rendered the SPRY domain versatile binding specificities and broad biological functions. These results serve as a useful basis for a profound characterization of the molecular interactions of SPRY7.

Fbxo45 Binds SPRY Motifs in the Extracellular Domain of N-Cadherin and Regulates Neuron Migration during Brain Development.

Several events during the normal development of the mammalian neocortex depend on N-cadherin, including the radial migration of immature projection neurons into the cortical plate. Remarkably, radial migration requires the N-cadherin extracellular domain but not N-cadherin-dependent homophilic cell-cell adhesion, suggesting that other N-cadherin-binding proteins may be involved. We used proximity ligation and affinity purification proteomics to identify N-cadherin-binding proteins. Both screens detected MycBP2 and SPRY domain protein Fbxo45, two components of an intracellular E3 ubiquitin ligase. Fbxo45 appears to be secreted by a nonclassical mechanism, not involving a signal peptide and not requiring transport from the endoplasmic reticulum to the Golgi apparatus. Fbxo45 binding requires N-cadherin SPRY motifs that are not involved in cell-cell adhesion. SPRY mutant N-cadherin does not support radial migration in vivo Radial migration was similarly inhibited when Fbxo45 expression was suppressed. The results suggest that projection neuron migration requires both Fbxo45 and the binding of Fbxo45 or another protein to SPRY motifs in the extracellular domain of N-cadherin.

The effector GpRbp-1 of Globodera pallida targets a nuclear HECT E3 ubiquitin ligase to modulate gene expression in the host.

Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

TRIM62-mediated restriction of avian leukosis virus subgroup J replication is dependent on the SPRY domain.

Emerging evidence suggests that some members of the tripartite motif (TRIM) family play a crucial role in antiretroviral. However, the chicken TRIM62 antiretroviral activity is unknown. Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus mainly inducing tumor formation and immunosuppression. The purpose of the study was to explore chicken TRIM62's role in ALV-J replication. In this study, we first tested the RNA expression of ALV-J and TRIM62 in chicken embryo fibroblasts (CEFs) cells infected with ALV-J by qRT-PCR. The result showed that ALV-J infection affected TRIM62 RNA expression, first upregulation and then downregulation, with the time course infection of ALV-J. Then, we silenced and overexpressed the TRIM62 to evaluate the effect of TRIM62 on ALV-J replication by qRT-PCR. We found that the knockdown of TRIM62 in CEF cells with shRNA targeting SPRY domain enhanced the viral replication more significantly than that with shRNA targeting coiled coil/unstructured domain, and overexpression of TRIM62 inhibited the viral replication. Further, we detected the effect of the domain deletion on TRIM62's antiviral activity. The result demonstrated that deletion of RING, B-box, coiled-coil domains partially abolished TRIM62's antiviral activity, while SPRY domain deletion resulted in the disappearance of antiviral activity of TRIM62. Taken together, our findings strongly suggested that TRIM62 plays an important role in the restriction of ALV-J replication, and SPRY domain is a prerequisite for the antiviral activity of TRIM62.

Crystal structure of the SPRY domain of human SPSB2 in the apo state.

The SPRY domain-containing SOCS box protein 2 (SPSB2) is one of four mammalian SPSB proteins that are characterized by a C-terminal SOCS box and a central SPRY/B30.2 domain. SPSB2 interacts with inducible nitric oxide synthase (iNOS) via the SPRY domain and polyubiquitinates iNOS, resulting in its proteasomal degradation. Inhibitors that can disrupt SPSB2-iNOS interaction and augment NO production may serve as novel anti-infective and anticancer agents. The previously determined murine SPSB2 structure may not reflect the true apo conformation of the iNOS-binding site. Here, the crystal structure of human SPSB2 SPRY domain in the apo state is reported at a resolution of 1.9 Å. Comparison of the apo and ligand-bound structures reveals that the iNOS-binding site is highly preformed and that major conformational changes do not occur upon ligand binding. Moreover, the C-terminal His6 tag of the recombinant protein binds to a shallow pocket adjacent to the iNOS-binding site on a crystallographically related SPSB2 molecule. These findings may help in structure-based and fragment-based SPSB2 inhibitor design in the future.

Critical Roles for Coiled-Coil Dimers of Butyrophilin 3A1 in the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T Cells.

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and tumors. Their TCRs respond to the sensing of isoprenoid metabolites, such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and isopentenyl pyrophosphate, by butyrophilin (BTN) 3A1. BTN3A1 is an Ig superfamily protein with extracellular IgV/IgC domains and intracellular B30.2 domains that bind prenyl pyrophosphates. We have proposed that intracellular α helices form a coiled-coil dimer that functions as a spacer for the B30.2 domains. To test this, five pairs of anchor residues were mutated to glycine to destabilize the coiled-coil dimer. Despite maintaining surface expression, BTN3A1 mutagenesis either abrogated or decreased stimulation by (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. BTN3A2 and BTN3A3 proteins and orthologs in alpacas and dolphins are also predicted to have similar coiled-coil dimers. A second short coiled-coil region dimerizes the B30.2 domains. Molecular dynamics simulations predict that mutation of a conserved tryptophan residue in this region will destabilize the dimer, explaining the loss of stimulation by BTN3A1 proteins with this mutation. The juxtamembrane regions of other BTN/BTN-like proteins with B30.2 domains are similarly predicted to assume α helices, with many predicted to form coiled-coil dimers. An exon at the end of this region and the exon encoding the dimerization region for B30.2 domains are highly conserved. We propose that coiled-coil dimers function as rod-like helical molecular spacers to position B30.2 domains, as interaction sites for other proteins, and as dimerization regions to allow sensing by B30.2 domains. In these ways, the coiled-coil domains of BTN3A1 play critical roles for its function.

Backbone 1H, 13C, and 15N resonance assignments of the PRY-SPRY domain of RNF135.

RING finger protein 135 (RNF135, also named Riplet or REUL) exerts multiple biological functions and its C-terminal PRY-SPRY/B30.2 domain is indispensable for most of these functions. RNF135 interacts with RIG-I (retinoic acid-inducible gene-I) via the PRY-SPRY domain and ubiquitinates RIG-I to promote innate anti-viral signaling, while mutations in the RNF135 gene can cause the Macrocephaly, macrosomia, facial dysmorphism (MMFD) syndrome, and RNF135 reportedly regulates the proliferation of glioblastoma cells as well as tongue cancer cells. Nevertheless, structure of full-length RNF135 or its PRY-SPRY domain has not been determined, and structural basis for molecular interactions involving RNF135 is largely unknown. Here we report the backbone 1H, 13C, and 15N chemical shift assignments of the PRY-SPRY domain of RNF135 and the secondary structure elements predicted based on chemical shifts, as well as the perturbations caused by the R286H mutation that is associated with MMFD syndrome. We found that the mutation did not alter the gross structure of the PRY-SPRY domain, so it may have impaired RNF135 function by affecting protein-protein interactions mediated by the domain.

The TRIM14 PRYSPRY domain mediates protein interaction via its basic interface.

Tripartite motif (TRIM)14 was recently shown to be an important molecule against pathogens. Its PRYSPRY domain acts as a protein-protein interaction module. TRIM14 exerts distinct functions via its PRYSPRY domain by interacting with different partners. However, the structural basis for its binding specificity remains unknown. Here we solved the crystal structure of the TRIM14 PRYSPRY domain, and found a positively charged surface that may mediate its partner specificity. Isothermal titration calorimetry reveals that the TRIM14 PRYSPRY domain binds to acidic peptides, and the analysis of the reported partners of TRIM14 is consistent with our assumption. Therefore, we demonstrate that the PRYSPRY domain of TRIM14 harbors a putative basic interface that may favorably bind to acidic amino acid residues.

A SPRY domain-containing SOCS box protein 3 (SPSB3) involved in the regulation of cytokine production in granulocytes of Crassostrea gigas.

The sp1A/ryanodine receptor (SPRY) family members have been reported to involve in important biological pathways, including innate immune signaling, cytokine signaling suppression, development, cell growth, and retroviral restriction. In the present study, a SPRY domain-containing SOCS box protein (named as CgSPSB3) was identified and characterized from oyster Crassostrea gigas. The open reading frame of CgSPSB3 gene was of 699 bp, encoding a polypeptide of 232 amino acid residues with a central SPRY domain and a C-terminal SOCS box motif. CgSPSB3 mRNA transcripts could be detected in all the examined tissues with the highest level in hemocytes, which was about 82.72-fold (p < 0.05) of that in gonad. Furthermore, the expression level of CgSPSB3 mRNA in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, which was about 2.04-fold (p < 0.05) of the average level of hemocytes. Immunofluorescence assay further revealed that CgSPSB3 protein was mainly distributed in the cytoplasm of granulocytes. The mRNA expression of CgSPSB3 in hemocytes was up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The mRNA expression of CgIFNLP, CgIL17-5 and CgTNF-1 decreased significantly (p < 0.05) at 24 h after the CgSPSB3 mRNA was knocked down by RNAi. These results collectively indicated that CgSPSB3 might play an important role in regulating cytokines production in granulocytes of C. gigas.

Ras enhances TGF-ß signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1.

BACKGROUND: Transformation by oncogene Ras overcomes TGF-ß mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial. METHODS: Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-ß receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-ß signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion. RESULTS: Ras interacts with the SPSB1's SPRY domain to enhance TGF-ß signaling. Ras interacts and colocalizes with the TGF-ß type II receptor's (TßRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TßRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-ß signaling and its mediated migration and invasion. CONCLUSION: Ras positively cooperates with TGF-ß signaling by reducing the cellular protein levels of TßRII negative regualtor SPSB1.

Complex formation dynamics of native and mutated pyrin's B30.2 domain with caspase-1.

Pyrin protein is the product of the MEFV gene, mutations in which cause manifestation of familial Mediterranean fever (FMF). Functions of pyrin are not completely clear. The secondary structure of the pyrin is represented with four domains and two motifs. Mutations p.M680I, p.M694V, p.M694I, p.K695R, p.V726A, and p.A744S, which are located in the B30.2 domain of pyrin protein, are responsible for manifestation of the most common and severe forms of FMF. All the domains and the motifs of pyrin, are directly or indirectly, involved in the protein-protein interaction with proteins of apoptosis and regulate the cascade of inflammatory reactions, which is impaired due to pyrin mutations. It is well known, that malfunction of the pyrin-caspase-1 complex is the main reason of inflammation during FMF. Complete tertiary structure of pyrin and the effects of mutations in it are experimentally not studied yet. The aim of this study was to identify possible effects of the abovementioned mutations in the B30.2 domain tertiary structure and to determine their potential consequences in formation of the B30.2-caspase-1 complex. Using in silico methods, it was found, that these mutations led to structural rearrangements in B30.2 domain tertiary structure, causing shifts of binding sites and altering the interaction energy between B30.2 and caspase-1.

Rhesus monkey TRIM5α protein SPRY domain contributes to AP-1 activation.

TRIM5α is an important host restriction factor that could potently block retrovirus infection. The SPRY domain of TRIM5α mediates post-entry restriction by recognition of and binding to the retroviral capsid. Human TRIM5α also functions as an innate immune sensor to activate AP-1 and NF-κB signaling, which subsequently restrict virus replication. Previous studies have shown that the AP-1 and NF-κB signaling activation relies on the RING motif of TRIM5α. In this study, we have demonstrated that the SPRY domain is essential for rhesus macaque TRIM5α to activate AP-1 but not NF-κB signaling. The AP-1 activation mainly depends on all of the ß-sheet barrel on SPRY structure of TRIM5α. Furthermore, the SPRY-mediated auto-ubiquitination of TRIM5α is required for AP-1 activation. This study reports that rhesus macaque TRIM5α mainly undergoes Lys27-linked and Met1-linked auto-polyubiquitination. Finally, we found that the TRIM5α signaling function was positively correlated with its retroviral restriction activity. This study discovered an important role of the SPRY domain in immune signaling and antiviral activity and further expanded our knowledge of the antiviral mechanism of TRIM5α.

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.