Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

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.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Novel multivalent S100A8 inhibitory peptides attenuate tumor progression and metastasis by inhibiting the TLR4-dependent pathway.

The tumor-elicited inflammation is closely related to tumor microenvironment during tumor progression. S100A8, an endogenous ligand of Toll-like receptor 4 (TLR4), is known as a key molecule in the tumor microenvironment and premetastatic niche formation. We firstly generated a novel multivalent S100A8 competitive inhibitory peptide (divalent peptide3A5) against TLR4/MD-2, using the alanine scanning. Divalent peptide3A5 suppressed S100A8-mediated interleukin-8 and vascular endothelial growth factor production in human colorectal tumor SW480 cells. Using SW480-transplanted xenograft models, divalent peptide3A5 suppressed tumor progression in a dose-dependent manner. We demonstrated that combination therapy with divalent peptide3A5 and bevacizumab synergistically suppressed tumor growth in SW480 xenograft models. Using syngeneic mouse models, we found that divalent peptide3A5 improved the efficacy of anti-programmed death (PD)1 antibody, and lung metastasis. In addition, by using multivalent peptide library screening based on peptide3A5, we then isolated two more candidates; divalent ILVIK, and tetravalent ILVIK. Of note, multivalent ILVIK, but not monovalent ILVIK showed competitive inhibitory activity against TLR4/MD-2 complex, and anti-tumoral activity in SW480 xenograft models. As most tumor cells including SW480 cells also express TLR4, S100A8 inhibitory peptides would target both the tumor microenvironment and tumor cells. Thus, multivalent S100A8 inhibitory peptides would provide new pharmaceutical options for aggressive cancers.

TLR3 forms a laterally aligned multimeric complex along double-stranded RNA for efficient signal transduction.

Toll-like receptor 3 (TLR3) is a member of the TLR family, which plays an important role in the innate immune system and is responsible for recognizing viral double-stranded RNA (dsRNA). Previous biochemical and structural studies have revealed that a minimum length of approximately 40-50 base pairs of dsRNA is necessary for TLR3 binding and dimerization. However, efficient TLR3 activation requires longer dsRNA and the molecular mechanism underlying its dsRNA length-dependent activation remains unknown. Here, we report cryo-electron microscopy analyses of TLR3 complexed with longer dsRNA. TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction.

Activation and regulation mechanisms of NOD-like receptors based on structural biology.

Innate immunity is a primary defense system against microbial infections. Innate immune pattern recognition receptors (PRRs) play pivotal roles in detection of invading pathogens. When pathogens, such as bacteria and viruses, invade our bodies, their components are recognized by PRRs as pathogen-associated molecular patterns (PAMPs), activating the innate immune system. Cellular components such as DNA and RNA, acting as damage-associated molecular patterns (DAMPs), also activate innate immunity through PRRs under certain conditions. Activation of PRRs triggers inflammatory responses, interferon-mediated antiviral responses, and the activation of acquired immunity. Research on innate immune receptors is progressing rapidly. A variety of these receptors has been identified, and their regulatory mechanisms have been elucidated. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) constitute a major family of intracellular PRRs and are involved in not only combating pathogen invasion but also maintaining normal homeostasis. Some NLRs are known to form multi-protein complexes called inflammasomes, a process that ultimately leads to the production of inflammatory cytokines and induces pyroptosis through the proteolytic cascade. The aberrant activation of NLRs has been found to be associated with autoimmune diseases. Therefore, NLRs are considered targets for drug discovery, such as for antiviral drugs, immunostimulants, antiallergic drugs, and autoimmune disease drugs. This review summarizes our recent understanding of the activation and regulation mechanisms of NLRs, with a particular focus on their structural biology. These include NOD2, neuronal apoptosis inhibitory protein (NAIP)/NLRC4, NLR family pyrin domain containing 1 (NLRP1), NLRP3, NLRP6, and NLRP9. NLRs are involved in a variety of diseases, and their detailed activation mechanisms based on structural biology can aid in developing therapeutic agents in the future.

Structure of SARS-CoV-2 membrane protein essential for virus assembly.

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.

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 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.

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.

The structure of NLRP9 reveals a unique C-terminal region with putative regulatory function.

Nucleotide-binding and oligomerisation domain-like receptors (NLRs) can form inflammasomes that activate caspase-1 and pro-interleukin-1ß and induce pyroptosis. NLR family pyrin domain-containing 9 (NLRP9) forms an inflammasome and activates innate immune responses during virus infection, but little is known about this process. Here, we report the crystal and cryo-electron microscopy structures of NLRP9 in an ADP-bound state, revealing inactive and closed conformations of NLRP9 and its similarities to other structurally characterised NLRs. Moreover, we found a C-terminal region interacting with the concave surface of the leucine-rich repeat domain of NLRP9. This region is unique among NLRs and might be involved in the specific function of NLRP9. These data provide the structural basis for understanding the mechanism of NLRP9 regulation and activation.

Tetrasubstituted imidazoles as incognito Toll-like receptor 8 a(nta)gonists.

Small-molecule modulators of TLR8 have drawn much interests as it plays pivotal roles in the innate immune response to single-stranded RNAs (ssRNAs) derived from viruses. However, their clinical uses are limited because they can invoke an uncontrolled, global inflammatory response. The efforts described herein culminate in the fortuitous discovery of a tetrasubstituted imidazole CU-CPD107 which inhibits R848-induced TLR8 signaling. In stark contrast, CU-CPD107 shows unexpected synergistic agonist activities in the presence of ssRNA, while CU-CPD107 alone is unable to influence TLR8 signaling. CU-CPD107's unique, dichotomous behavior sheds light on a way to approach TLR agonists. CU-CPD107 offers the opportunity to avoid the undesired, global inflammation side effects that have rendered imidazoquinolines clinically irrelevant, providing an insight for the development of antiviral drugs.

Improving particle quality in cryo-EM analysis using a PEGylation method.

Cryo-electron microscopy (cryo-EM) is widely used for structural biology studies and has been developed extensively in recent years. However, its sample vitrification process is a major limitation because it causes severe particle aggregation and/or denaturation. This effect is thought to occur because particles tend to stick to the "deadly" air-water interface during vitrification. Here, we report a method for PEGylation of proteins that can efficiently protect particles against such problems during vitrification. This method alleviates the laborious process of fine-tuning the vitrification conditions, allowing for analysis of samples that would otherwise be discarded.

Cryo-EM structures of Toll-like receptors in complex with UNC93B1.

Nucleic acid-sensing Toll-like receptors (TLRs) play a pivotal role in innate immunity by recognizing foreign DNA and RNA. Compartmentalization of these TLRs in the endosome limits their activation by self-derived nucleic acids and reduces the possibility of autoimmune reactions. Although chaperone Unc-93 homolog B1, TLR signaling regulator (UNC93B1) is indispensable for the trafficking of TLRs from the endoplasmic reticulum to the endosome, mechanisms of UNC93B1-mediated TLR regulation remain largely unknown. Here, we report two cryo-EM structures of human and mouse TLR3-UNC93B1 complexes and a human TLR7-UNC93B1 complex. UNC93B1 exhibits structural similarity to the major facilitator superfamily transporters. Both TLRs interact with the UNC93B1 amino-terminal six-helix bundle through their transmembrane and luminal juxtamembrane regions, but the complexes of TLR3 and TLR7 with UNC93B1 differ in their oligomerization state. The structural information provided here should aid in designing compounds to combat autoimmune diseases.

Structural analysis reveals TLR7 dynamics underlying antagonism.

Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. These compounds protect mice against lethal autoimmunity. Combining crystallography and cryo-electron microscopy, we identify the open conformation of the receptor and reveal the structural equilibrium between open and closed conformations that underlies TLR7 antagonism, as well as the detailed mechanism by which TLR7-specific antagonists bind to their binding pocket in TLR7. Our work provides small-molecule TLR7-specific antagonists and suggests the TLR7-targeting strategy for treating autoimmune diseases.

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.

Crystal structure of the FYCO1 RUN domain suggests possible interfaces with small GTPases.

FYCO1 is a multidomain adaptor protein that plays an important role in autophagy by mediating the kinesin-dependent microtubule plus-end-directed transport of autophagosomes. FYCO1 contains a RUN domain, which is hypothesized to function as a specific effector for members of the Ras superfamily of small GTPases, but its role has not been well characterized and its interaction partner(s) have not been identified. Here, the crystal structure of the FYCO1 RUN domain was determined at 1.3 Šresolution. The overall structure of the FYCO1 RUN domain was similar to those of previously reported RUN domains. Detailed structural comparisons with other RUN domains and docking studies suggested a possible interaction interface of the FYCO1 RUN domain with small GTPases of the Ras superfamily.

Crystal structure of TEX101, a glycoprotein essential for male fertility, reveals the presence of tandemly arranged Ly6/uPAR domains.

Testis-expressed gene 101 (TEX101) is a glycosyl-phosphatidylinositol-anchored glycoprotein essential for sperm fertility and spermatogenesis. TEX101 interacts with lymphocyte antigen 6 complex, locus K (Ly6k) as well as a disintegrin and metallopeptidase domain 3 (ADAM3). Although these proteins are considered essential for fertility, the associated mechanisms remain uncharacterized. Herein, we determined the crystal structure of human and mouse TEX101, revealing that TEX101 contains two tandem Ly6/uPAR (LU) domains. Detailed structural analyses revealed characteristic surfaces of TEX101 that may be involved in the interactions with other proteins or membranes. These results provide the structural basis for the role of TEX101 in fertilization and could contribute to developing diagnostic methods and treatments for infertility or developing male contraceptives.

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.

Rationally Designed Small-Molecule Inhibitors Targeting an Unconventional Pocket on the TLR8 Protein-Protein Interface.

Rational designs of small-molecule inhibitors targeting protein-protein interfaces have met little success. Herein, we have designed a series of triazole derivatives with a novel scaffold to specifically intervene with the interaction of TLR8 homomerization. In multiple assays, TH1027 was identified as a highly potent and specific inhibitor of TLR8. A successful solution of the X-ray crystal structure of TLR8 in complex with TH1027 provided an in-depth mechanistic insight into its binding mode, validating that TH1027 was located between two TLR8 monomers and recognized as an unconventional pocket, thereby preventing TLR8 from activation. Further biological evaluations showed that TH1027 dose-dependently suppressed the TLR8-mediated inflammatory responses in both human monocyte cell lines, peripheral blood mononuclear cells, and rheumatoid arthritis patient specimens, suggesting a strong therapeutic potential against autoimmune diseases.