Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

Structure of the bile acid transporter and HBV receptor NTCP.

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.

Toll-like Receptor 9 Contains Two DNA Binding Sites that Function Cooperatively to Promote Receptor Dimerization and Activation.

Toll-like receptor 9 (TLR9) recognizes DNA containing CpG motifs derived from bacteria and viruses and activates the innate immune response to eliminate them. TLR9 is known to bind to CpG DNA, and here, we identified another DNA binding site in TLR9 that binds DNA containing cytosine at the second position from the 5' end (5'-xCx DNA). 5'-xCx DNAs bound to TLR9 in the presence of CpG DNA and cooperatively promoted dimerization and activation of TLR9. Binding at both sites was important for efficient activation of TLR9. The 5'-xCx DNA bound the site corresponding to the nucleoside binding site in TLR7 and TLR8 as revealed by the structural analysis. This study revealed that TLR9 recognizes two types of DNA through its two binding sites for efficient activation. This information may contribute to the development of drugs that control the activity of TLR9.

Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA.

Toll-like receptor 7 (TLR7) is a single-stranded RNA (ssRNA) sensor in innate immunity and also responds to guanosine and chemical ligands, such as imidazoquinoline compounds. However, TLR7 activation mechanism by these ligands remain largely unknown. Here, we generated crystal structures of three TLR7 complexes, and found that all formed an activated m-shaped dimer with two ligand-binding sites. The first site conserved in TLR7 and TLR8 was used for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 was used for a ssRNA-binding that enhanced the affinity of the first-site ligands. The first site preferentially recognized guanosine and the second site specifically bound to uridine moieties in ssRNA. Our structural, biochemical, and mutagenesis studies indicated that TLR7 is a dual receptor for guanosine and uridine-containing ssRNA. Our findings have important implications for understanding of TLR7 function, as well as for therapeutic manipulation of TLR7 activation.

Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation.

SignificanceThe nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that forms an inflammasome. The cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950 has established the structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation and the mechanism of action of the NLRP3 specific inhibitor. The inactive NLRP3 oligomer represents the NLRP3 resting state, capable of binding to membranes and is likely disrupted for its activation. Visualization of the inhibitor binding mode will enable optimization of the activity of NLRP3 inflammasome inhibitor drugs.

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

Propofol directly binds to and inhibits TLR7.

Sedatives/anesthetics are important medical tools to facilitate medical care and increase patients' comfort. Increasingly, there is recognition that sedatives/anesthetics can modulate immune functions. Toll-like receptors (TLRs) are major pattern recognition receptors involved in the recognition of microbial components. TLR7 recognizes single-strand RNA virus such as influenza and SARS-CoV2 viruses and initiates interferon (IFN) responses. IFN production triggered by TLR7 stimulation is a critical anti-viral response. For example, patients with TLR7 variants including loss-of- function variants were associated with severe COVID-19. Taken together, it is important to determine if sedatives/anesthetics mitigate TLR7 function. We have previously showed that TLR7-mediated activation was not affected by volatile anesthetics. However, we found that propofol attenuated TLR7 activation among intravenous sedatives in the reporter assay. TLR7 agonist R837 stimulation increased TNF-α, IL-1ß, IL-6, IL-10, and IFN-ß mRNA levels in bone marrow-derived dendritic cells, while these levels were attenuated by propofol. Our murine lung slice experiments showed that propofol attenuated IFN production. R837 increased IFN-ß expression in the lungs, and propofol attenuated IFN-ß expression in an in vivo model of R837 intranasal instillation. We also found that propofol directly bound to and hindered its association of TLR7 with MyD88. Our analysis using fropofol, propofol derivative showed that the hydroxyl group in propofol was important for propofol-TLR7 interaction.

Structure of IZUMO1-JUNO reveals sperm-oocyte recognition during mammalian fertilization.

Fertilization is a fundamental process in sexual reproduction, creating a new individual through the combination of male and female gametes. The IZUMO1 sperm membrane protein and its counterpart oocyte receptor JUNO have been identified as essential factors for sperm-oocyte interaction and fusion. However, the mechanism underlying their specific recognition remains poorly defined. Here, we show the crystal structures of human IZUMO1, JUNO and the IZUMO1-JUNO complex, establishing the structural basis for the IZUMO1-JUNO-mediated sperm-oocyte interaction. IZUMO1 exhibits an elongated rod-shaped structure comprised of a helical bundle IZUMO domain and an immunoglobulin-like domain that are each firmly anchored to an intervening ß-hairpin region through conserved disulfide bonds. The central ß-hairpin region of IZUMO1 provides the main platform for JUNO binding, while the surface located behind the putative JUNO ligand binding pocket is involved in IZUMO1 binding. Structure-based mutagenesis analysis confirms the biological importance of the IZUMO1-JUNO interaction. This structure provides a major step towards elucidating an essential phase of fertilization and it will contribute to the development of new therapeutic interventions for fertility, such as contraceptive agents.

The effect of anesthetics on toll like receptor 9.

Toll like receptors (TLRs) are critical receptors to respond to danger signals, and their functions are relevant in the perioperative period. We previously reported that volatile anesthetics directly bound to TLR2 and TLR4 and attenuated their functions. Given that TLR9 can respond to mitochondrial DNA, a danger signal that is released upon tissue injury, we examined the role of anesthetics on TLR9 function. Our reporter assay showed that volatile anesthetics isoflurane and sevoflurane increased the activation of TLR9, while propofol attenuated it. TLR9 activation occurs via its dimerization. The dimerization is facilitated by unmethylated cytosine-phosphate-guanine (CpG) DNA as well as DNA containing cytosine at the second position from 5'-end (5'-xCx DNA). Our structural analysis using photoactivable anesthetics and rigid docking simulation showed that isoflurane and sevoflurane bound to both TLR9 dimer interface and 5'-xCx DNA binding site. Propofol bound to the TLR9 antagonist binding site. This is the first illustration that anesthetics can affect the binding of nucleic acids to their receptor. This study sets the foundation for the effect of anesthetics on TLR9 and will pave the way for future studies to determine the significance of such interactions in the clinical setting.

Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9.

Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses. TLR9 recognizes bacterial and viral DNA containing the cytosine-phosphate-guanine (CpG) dideoxynucleotide motif. The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9-CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT-LRR10) from one protomer and the carboxy-terminal fragment (LRR20-LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2-LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9.

Small-molecule inhibition of TLR8 through stabilization of its resting state.

Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and DNA molecular patterns. Nonetheless, few small molecules can selectively modulate these TLRs. In this manuscript, we identified the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. As a proof of concept of their therapeutic potential, we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.

Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.

General anesthesia has been the requisite component of surgical procedures for over 150 yr. Although immunomodulatory effects of volatile anesthetics have been growingly appreciated, the molecular mechanism has not been understood. In septic mice, the commonly used volatile anesthetic isoflurane attenuated the production of 5-lipoxygenase products and IL-10 and reduced CD11b and intercellular adhesion molecule-1 expression on neutrophils, suggesting the attenuation of TLR4 signaling. We confirmed the attenuation of TLR4 signaling in vitro and their direct binding to TLR4-myeloid differentiation-2 (MD-2) complex by photolabeling experiments. The binding sites of volatile anesthetics isoflurane and sevoflurane were located near critical residues for TLR4-MD-2 complex formation and TLR4-MD-2-LPS dimerization. Additionally, TLR4 activation was not attenuated by intravenous anesthetics, except for a high concentration of propofol. Considering the important role of TLR4 system in the perioperative settings, these findings suggest the possibility that anesthetic choice may modulate the outcome in patients or surgical cases in which TLR4 activation is expected.-Okuno, T., Koutsogiannaki, S., Hou, L., Bu, W., Ohto, U., Eckenhoff, R. G., Yokomizo, T., Yuki, K. Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.

Mechanisms controlling nucleic acid-sensing Toll-like receptors.

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) respond to DNA/RNA derived from pathogens and dead cells. Structural studies have revealed a variety of molecular mechanisms by which TLRs sense NAs. Double-stranded RNA and single-stranded DNA directly bind to TLR3 and TLR9, respectively, whereas TLR7 and TLR8 bind to nucleosides and oligoribonucleotides derived from RNAs. Activation of ligand-bound TLRs is influenced by the functional status of TLRs. Proteolytic cleavage of NA-sensing TLRs enables ligand-dependent TLR dimerization. Trafficking of ligand-activated TLRs in endosomal and lysosomal compartments is requisite for production of type I interferons. Activation of NA-sensing TLRs is required for the control of viruses such as herpes simplex virus and endogenous retroviruses. On the other hand, excessive activation of NA-sensing TLRs drives disease progression in a variety of inflammatory diseases including systemic lupus erythematosus, heart failure, arthritis and non-alcoholic steatohepatitis. NA-sensing TLRs are targets for therapeutic intervention in these diseases. We here focus on our recent progresses in our understanding of NA-sensing TLRs.

Autoinhibition and relief mechanism by the proteolytic processing of Toll-like receptor 8.

Toll-like receptor 8 (TLR8) senses single-stranded RNA (ssRNA) and initiates innate immune responses. TLR8 requires proteolytic cleavage at the loop region (Z-loop) between leucine-rich repeat (LRR) 14 and LRR15 for its activation. However, the molecular basis of Z-loop processing remains unknown. To elucidate the mechanism of Z-loop processing, we performed biochemical and structural studies of how the Z-loop affects the function of TLR8. TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. Crystallographic analysis revealed that the uncleaved Z-loop located on the ascending lateral face prevents the approach of the dimerization partner by steric hindrance. This autoinhibition mechanism of dimerization by the Z-loop might be occurring in the proteins of the same subfamily, TLR7 and TLR9.

The crystal structure of the AhRR-ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR-ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR-ARNT and AhR-ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR-ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR-ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription.

Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-ß expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.

Guanosine and its modified derivatives are endogenous ligands for TLR7.

Toll-like receptor (TLR) 7and 8 were considered to recognize single-strand RNA (ssRNA) from viruses. Although these receptors also respond to synthetic small chemical ligands, such as CL075 and R848, it remains to be determined whether these receptors sense natural small molecules or not. In the structure of human TLR8 (huTLR8) with ssRNA, there are two ligand-binding sites: one binds a uridine and the other binds an oligoribonucleotide (ORN). This finding demonstrates that huTLR8 recognizes degradation products of ssRNA, suggesting the presence of natural small ligands. We here show that TLR7 works as the sensor for guanosine (G)/2'-deoxyguanosine (dG) in the presence of ORN where ORN strengthens TLR7 interaction with G/dG. In addition, modified nucleosides such as 7-methylguanosine, 8-hydroxyguanosine (8-OHG) and 8-hydroxydeoxyguanosine (8-OHdG) activated TLR7 with ORNs. Importantly, 8-OHdG-a well-known oxidative DNA damage marker with unknown function-induced strong cytokine production comparable to G and dG both in mouse and human immune cells. Although 8-OHdG bound TLR7/ORN with lower affinity than dG did in isothermal titration calorimetry, administered 8-OHdG was metabolically more stable than dG in the serum, indicating that 8-OHdG acts on TLR7 as an endogenous ligand in vivo To address a role of G analogs in the disease state, we also examined macrophages from Unc93b1 (D34A/D34A) mice, which suffer from TLR7-dependent systemic inflammation, and found that Unc93b1 (D34A/D34A) macrophages showed significantly enhanced response to G alone or 8-OHdG with ORN. In conclusion, our results provide evidence that G, dG, 8-OHG and 8-OHdG are novel endogenous ligands for TLR7.

Conservation and Divergence of Ligand Recognition and Signal Transduction Mechanisms in Toll-Like Receptors.

Toll-like receptors (TLRs) play a central role in innate immunity as pathogen sensors. During the last decade, structural analyses of TLRs have revealed the mechanisms of ligand recognition and signal transduction. Each TLR recognizes its cognate ligand in a different manner, whereas signal transduction is achieved by a common mechanism. In this review, the mechanisms of ligand recognition and signal transduction by TLRs are summarized based on recent structural information.

TLR4-MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide.

Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4-MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4-MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4-MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4-MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.

Structural basis of pharmacological chaperoning for human ß-galactosidase.

GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal ß-galactosidase (ß-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human ß-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, ß-Gal(R201C) and ß-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of ß-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of ß-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of ß-Gal selective chaperoning by newly developed PC compounds.