Umeharu Ohto

Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa

Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD-2 and Toll-like receptor 4. Crystal structures of human MD-2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS have been determined at 2.0 and 2.2 angstrom resolutions, respectively. MD-2 shows a deep hydrophobic cavity sandwiched by two β sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. These structures suggest that MD-2 plays a principal role in endotoxin recognition and provide a basis for antiseptic drug development.

Structural Reorganization of the Toll-Like Receptor 8 Dimer Induced by Agonistic Ligands

Dissecting TLR8 Interactions Toll-like receptors (TLRs) activate the innate immune system in response to invading pathogens. TLR7 and TLR8 recognize single-stranded RNA from viruses and also contribute to the pathogenesis of autoimmune diseases. Tanji et al. (p. 1426) now report the crystal structure of the unliganded TLR8 ectodomain and the TLR8 ectodomain bound to three different small-molecule agonists. Ligand binding to preformed TLR8 dimers induced conformational changes that brought the C-terminal domains closer together, presumably initiating downstream signaling. The crystal structure of unbound and ligand-bound Toll-like receptor 8 reveals ligand-induced conformational changes. Toll-like receptor 7 (TLR7) and TLR8 recognize single-stranded RNA and initiate innate immune responses. Several synthetic agonists of TLR7-TLR8 display novel therapeutic potential; however, the molecular basis for ligand recognition and activation of signaling by TLR7 or TLR8 is largely unknown. In this study, the crystal structures of unliganded and ligand-induced activated human TLR8 dimers were elucidated. Ligand recognition was mediated by a dimerization interface formed by two protomers. Upon ligand stimulation, the TLR8 dimer was reorganized such that the two C termini were brought into proximity. The loop between leucine-rich repeat 14 (LRR14) and LRR15 was cleaved; however, the N- and C-terminal halves remained associated and contributed to ligand recognition and dimerization. Thus, ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes.

Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2

Lipopolysaccharide (LPS), also known as endotoxin, activates the innate immune response through toll-like receptor 4 (TLR4) and its coreceptor, MD-2. MD-2 has a unique hydrophobic cavity that directly binds to lipid A, the active center of LPS. Tetraacylated lipid IVa, a synthetic lipid A precursor, acts as a weak agonist to mouse TLR4/MD-2, but as an antagonist to human TLR4/MD-2. However, it remains unclear as to how LPS and lipid IVa show agonistic or antagonistic activities in a species-specific manner. The present study reports the crystal structures of mouse TLR4/MD-2/LPS and TLR4/MD-2/lipid IVa complexes at 2.5 and 2.7 Å resolutions, respectively. Mouse TLR4/MD-2/LPS exhibited an agonistic “m”-shaped 2:2:2 complex similar to the human TLR4/MD-2/LPS complex. Mouse TLR4/MD-2/lipid IVa complex also showed an agonistic structural feature, exhibiting architecture similar to the 2:2:2 complex. Remarkably, lipid IVa in the mouse TLR4/MD-2 complex occupied nearly the same space as LPS, although lipid IVa lacked the two acyl chains. Human MD-2 binds lipid IVa in an antagonistic manner completely differently from the way mouse MD-2 does. Together, the results provide structural evidence of the agonistic property of lipid IVa on mouse TLR4/MD-2 and deepen understanding of the ligand binding and dimerization mechanism by the structurally diverse LPS variants.

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.

Toll-like receptor 8 senses degradation products of single-stranded RNA

Toll-like receptor 8 (TLR8) recognizes viral or bacterial single-stranded RNA (ssRNA) and activates innate immune systems. TLR8 is activated by uridine- and guanosine-rich ssRNA as well as by certain synthetic chemicals; however, the molecular basis for ssRNA recognition has remained unknown. In this study, to elucidate the recognition mechanism of ssRNA, we determined the crystal structures of human TLR8 in complex with ssRNA. TLR8 recognized two degradation products of ssRNA—uridine and a short oligonucleotide—at two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. Site-directed mutagenesis revealed that both binding sites were essential for activation of TLR8 by ssRNA. These results demonstrate that TLR8 is a sensor for both uridine and a short oligonucleotide derived from RNA.

Coagulation Factor X Activates Innate Immunity to Human Species C Adenovirus

Wound Healing and Immunity Although wound healing and infection are often overlapping processes, whether the wound healing response modulates the immune response is not well understood. Doronin et al. (p. 795, published online 27 September; see the Perspective by Herzog and Ostrov) now show that coagulation factor X, an important component of the blood clotting cascade, helps to trigger antiviral immunity in response to adenovirus infection in mice. Factor X binds to human type C adenovirus with very high affinity. Structural analysis identified the critical binding residues between factor X and adenovirus, which, when mutated, inhibited binding. Despite being able to infect splenic macrophages in mice, transcriptional profiling of spleens from mice infected with a mutant adenovirus unable to bind to factor X revealed impaired activation of signaling cascades associated with innate immunity. Tagging adenovirus with a serum protein prompts an immune response when the virus enters cells. Although coagulation factors play a role in host defense for “living fossils” such as horseshoe crabs, the role of the coagulation system in immunity in higher organisms remains unclear. We modeled the interface of human species C adenovirus (HAdv) interaction with coagulation factor X (FX) and introduced a mutation that abrogated formation of the HAdv-FX complex. In vivo genome-wide transcriptional profiling revealed that FX-binding–ablated virus failed to activate a distinct network of nuclear factor κB–dependent early-response genes that are activated by HAdv-FX complex downstream of TLR4/MyD88/TRIF/TRAF6 signaling. Our study implicates host factor “decoration” of the virus as a mechanism to trigger an innate immune sensor that responds to a misplacement of coagulation FX from the blood into intracellular macrophage compartments upon virus entry into the cell.

Impaired α-TTP-PIPs Interaction Underlies Familial Vitamin E Deficiency

Vitamin E Out Familial vitamin E deficiency is caused by mutations in the α-tocopherol transfer protein (α-TTP) gene. Kono et al. (p. 1106, published online 18 April; see the Perspective by Mesmin and Antonny) studied natural mutations in α-TTP. α-TTP bound phosphatidylinositol polyphosphates (PIPs), especially PI(4,5)P2, and a disease-related missense mutation abolished PIP binding but not α-tocopherol binding. The x-ray crystal structure of the α-TTP–PIP complex suggested that PIP binding opens the lid of the α-tocopherol–binding pocket to facilitate the release of α-tocopherol. Thus, PIP binding to α-TTP at the target membrane may facilitate the release of α-tocopherol in the hydrophobic pocket of α-TTP to the lipid bilayer of the target membrane, providing a mechanism for the transfer of lipids from the lipid-transfer protein to the target membrane. Phosphatidylinositol phosphates may play a role in lipid-transfer protein–mediated vitamin E efflux from hepatocytes. [Also see Perspective by Mesmin and Antonny] α-Tocopherol (vitamin E) transfer protein (α-TTP) regulates the secretion of α-tocopherol from liver cells. Missense mutations of some arginine residues at the surface of α-TTP cause severe vitamin E deficiency in humans, but the role of these residues is unclear. Here, we found that wild-type α-TTP bound phosphatidylinositol phosphates (PIPs), whereas the arginine mutants did not. In addition, PIPs in the target membrane promoted the intermembrane transfer of α-tocopherol by α-TTP. The crystal structure of the α-TTP–PIPs complex revealed that the disease-related arginine residues interacted with phosphate groups of the PIPs and that the PIPs binding caused the lid of the α-tocopherol–binding pocket to open. Thus, PIPs have a role in promoting the release of a ligand from a lipid-transfer protein.

Crystal Structure of Human β-Galactosidase STRUCTURAL BASIS OF GM1 GANGLIOSIDOSIS AND MORQUIO B DISEASES

Background: Deficiencies in β-d-galactosidase cause lysosomal storage diseases. Results: This is the first report to describe the crystal structure of human β-Gal. Human β-Gal is composed of a TIM barrel domain and two β-domains. Conclusion: The mutations were classified as mutations directly affecting the ligand recognition, mutations inside the protein core, or mutations located in the protein surface. Significance: Structural insights into lysosomal storage diseases mutations can be demonstrated. GM1 gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, GM1 ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.

Structural analyses of human Toll-like receptor 4 polymorphisms D299G and T399I

Background: TLR4 polymorphism replacing Asp-299 with Gly and Thr-399 with Ile (D299G/T399I) causes LPS hyporesponsiveness. Results: TLR4SNPs·MD-2·LPS exhibits an agonistic 2:2:2 architecture. Local structural differences were observed around D299G, but not around T399I, SNP site. Conclusion: These local differences cause the modulation of surface properties of TLR4, which may affect ligand binding. Significance: This study provides structural evidence of the functionality of the mutant TLR4 carrying the SNPs. Toll-like receptor 4 (TLR4) and its coreceptor MD-2 recognize bacterial lipopolysaccharide (LPS) and signal the innate immune response. Two single nucleotide polymorphisms (SNPs) of human TLR4, D299G and T399I, have been identified and suggested to be associated with LPS hyporesponsiveness. Moreover, the SNPs have been proposed to be associated with a variety of infectious and noninfectious diseases. However, how the SNPs affect the function of TLR4 remains largely unknown. Here, we report the crystal structure of the human TLR4 (D299G/T399I)·MD-2·LPS complex at 2.4 Å resolution. The ternary complex exhibited an agonistic “m”-shaped 2:2:2 architecture that was similar to that of the human wild type TLR4·MD-2·LPS complex. Local structural differences that might affect the binding of the ligands were observed around D299G, but not around T399I, SNP site.

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.