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Review Article

Structural Characterization of Nucleic Acid-sensing Toll-like Receptors

The senses suggest the following mechanism stranded (ss)RNA, and TLR9 senses pathogen-associated or cell damage- of TLR activation. Inactive TLRs DNA with unmethylated cytosine associated structurally conserved exist as monomers, and ligand phosphate-guanosine (CpG) motifs molecules through various pattern binding induces dimerization of the (Fig. 2(b)). In addition, mouse TLR13 recognition receptors (PPRs) [1]. extracellular TLR domain, producing detects bacterial 23S ribosomal RNA. Nucleic acids that are released a typical “m”-shaped structure, where In contrast to other TLRs, TLR3 and from viruses, bacteria or dead cells the intracellular C-terminal regions of TLRs 7–9 are expressed on endosomal during infection or tissue damage the two TLR protomers are positioned membranes, limiting the recognition are principal ligands to PPRs. PRR in close proximity. Subsequent of self-derived ligands that are involved in nucleic acid recognition dimerization of the intracellular TIR released from dying cells (Fig. 1). In can be divided into two groups on the domain is followed by the recruitment addition, the members of the TLR7 basis of cellular localization: several of adaptor that execute signal subfamily comprising TLRs 7, 8, and members of membrane-bound Toll- transduction. 9 contain a characteristic long inserted like receptors (TLRs) and cytoplasmic Viral/bacterial nucleic acids are loop region (known as the Z-loop) PPRs such as Nod-like receptors potent stimulators of innate immunity. composed of approximately 30 amino (NLRs), RIG-I like receptors (RLRs), In humans, nearly half the TLRs acid residues, and localized in the and the DNA recognition recognize nucleic acid ligands. TLR3 region between LRR14 and LRR15 cyclic GMP-AMP synthase (cGAS). responds to double-stranded (ds) (Fig. 2(a,b)). Z-loop processing is TLRs, evolutionarily conserved RNA, while TLR7 and TLR8, which required for the activation of these membrane-spanning receptors are closely related, recognize single- TLRs. homologous to the Drosophila Toll , are mostly expressed in macrophages and dendritic cells. The recognition of pathogen-associated Triacyl Diacyl molecular patterns (PAMPs) or lipoprotein lipoprotein LPS Flagellin damage-associated molecular patterns CG MD-2 (DAMPs) by TLRs is critical for CpG DNA ssRNA dsRNA the activation of the transcription HQGRVRPH TLR2 TLR1 TLR2 TLR6 TLR4 TLR5 factor NF-κB or IRFs, leading to TLR10 unknown the production of pro-inflammatory or type I interferons (Fig. 1). MyD88 TLR9 TLRs are glycosylated type I integral TLR7 TLR3 TRIF IRF3 IRF7 NF-κB TLR8 membrane receptors with N-terminal Interferon extracellular leucine-rich repeats Inflammatory IRF3

(LRRs), a transmembrane domain, nucleus and a C-terminal cytoplasmic domain (Fig. 2(a)). The LRR domain contains binding sites for PAMPs or DAMPs, Fig. 1. Schematic illustration of human TLR signaling while the cytoplasmic domain, TLR signaling is initiated by ligand-induced dimerization of receptors, followed by the engagement of TIR-domain-containing adaptor known as the Toll/IL-1 receptor proteins, such as MyD88 and TRIF, which activate downstream (TIR) region, activates downstream signaling cascades. A major consequence of TLR signaling is the signaling cascades by interacting with induction of proinflammatory cytokines and type I interferons. TLR5 adaptor proteins such as MyD88 and and heterodimers of TLR2 and TLR1 or TLR6 are expressed at the cell surface, whereas TLR3 and TLR7–9 localize to the endosome, TRIF [2]. Biochemical and structural where they sense microbial and host-derived nucleic acids. TLR4 studies on TLR extracellular domains localizes to both the plasma membrane and the endosome.

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Although TLR7 and TLR8 Asp543 of TLR8; and (iii) snug fitting region distinct from that of TLR8 have been considered to primarily of two substituents of a ligand to the in its spatial position and ligand- recognize ssRNA, they are also small hydrophobic pocket formed recognition mode. As non-terminal activated by small synthetic ligands between the two protomers (Fig. 3(b)). uridine was specifically recognized such as imidazoquinolines and Next, the crystal structure of whereas other uridines were loosely nucleoside analogs, raising a question TLR8 complexed with 20-mer ssRNA recognized, diverse oligonucleotides concerning the molecular basis of was determined [5]. Unexpectedly, can be accomodated at the second the recognition of these structurally the crystal structure revealed that site as long as they contain uridine. and chemically distinct ligands. rather than binding directly to the Taken all together, TLR7 is a dual Furthermore, expression patterns of full-length ssRNA, TLR8 binds sensor for guanosine and uridine- TLR7 and TLR8 are different, but it to RNA degradation products at containing ssRNA, while TLR8 remained to be determined whether two distinct sites (Fig. 3(a)). The acts as a uridine sensor recognizing or not the biochemical features and first site, which accommodates the diverse nucleotide compositions of recognition mechanisms of TLR7 and ssRNA degradation product uridine, ssRNA. These findings indicate that TLR8 are different. Moreover, the is identical to a previously reported although TLR7 and TLR8 are closely mechanism of the recognition of a binding site for small synthetic related receptors and demonstrate pathogenic DNA sequence by TLR9 ligands [4]. The second binding site similar activation mechanisms, they has not been clarified in detail. located at the interior of the TLR8 have significant differences in ligand We used brilliant synchrotron ring structure is sandwiched between recognition patterns. Biochemical X-rays at SPring8 BL41XU and KEK the concave surface and the Z-loop analysis provided further insights to determine the crystal structures and holds ssRNA molecules longer into the ligand specificity of TLR7 of the LRR domains of TLR7 [3], than 2 nucleotides. Most recently, by showing that not only guanosine TLR8 [4,5] and TLR9 [6]. First, the structure of the activated TLR7- but also its modified derivatives the structures of unliganded and guanosine-ssRNA complex has (e.g., 7-methylguanosine and small synthetic ligand-bound TLR8 been determined [3] (Fig. 3(a)). 8-hydroxyguanosine) may serve as were determined (Fig. 3(a)) [4]. Analogous to the TLR8 structure, TLR7 endogenous ligands [7]. Chemical ligands bind to TLR8 which recognizes uridine and ssRNA Moreover, the research group at two equivalent positions on a at distinct sites, TLR7 interacts with clarified the crystal structure of three symmetrical 2:2 complex, triggering guanosine and polyU at two different forms of TLR9, namely, unliganded the rearrangement of the dimeric sites. The first site is identical to that TLR9, TLR9 bound to a CpG- configuration. Then, TLR8 C-termini of TLR8, and accommodates small containing DNA, and TLR9 bound are brought into closer proximity, agonists; however, it preferentially to an inhibitory DNA (iDNA) [6]. thus generating a more compact binds guanosine, while TLR8 CpG-containing DNA binds to TLR9 structure than that of the m-shaped accommodates uridine. The second at a stoichiometric ratio of 2:2 in dimer. The ligand is positioned at site presents an ssRNA-binding an extended conformation. CpG- the TLR8 dimer interface between the N-terminal half of one protomer and the C-terminal half of the other protomer, stabilizing the activated (a) (b) human Z-loop form of TLR8. Prior to structural Z-loop TLR7 812aa studies of TLRs, it was not known LRR ssRNA 40% (73%) whether the N-terminal fragment in (Leucine-Rich Z-loop Repeats) the TLR7 family was required for TLR8 801aa ssRNA 100% ligand recognition following Z-loop Membrane Z-loop Toll / IL-1 TLR9 789aa processing. However, structural Receptor (TIR) CpG-DNA 34% (66%) observations clearly demonstrated Domain Extracellular domain that both N- and C-terminal fragments of TLR8 are necessary for ligand Fig. 2. Schematic representation of nucleic acid-sensing TLRs recognition. Three key interactions (a) Schematic representation of the domain organization in nucleic are important in ligand recognition: acid-sensing TLRs. TLRs consist of an extracellular LRR domain, (i) stacking interactions between the a transmembrane domain, and a cytoplasmic TIR domain. The characteristic Z-loop is shown in red. (b) Schematic representation benzene ring of ligands and Phe405 of of the extracellular domains of nucleic acid-sensing TLRs. Sequence TLR8; (ii) hydrogen bonds between homology to TLR8 is shown, and numbers in parentheses are the the amidine group of ligands and values of sequence identity. The cognate ligands are also shown.

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exhibits a stronger binding affinity (a) TLR8/ssRNA uridine for TLR9 than do CpG-containing ssRNA(UG) ssRNA(UG) agonists, the overlap would account for the antagonistic effect of iDNA. Synergistic activation by two ligands, revealed by the structural works in TLR7 and TLR8, is a new N N concept of the activation mechanism CC TLR7_G_ssRNA guanosine [3,5]. Although TLR7 and TLR8 ssRNA(UUU) exhibited the preference for guanosine and uridine, respectively, over other mononucleosides or mononucleotides at the first binding site, the binding affinity to these ligands is still lower

ssRNA(UUU) N N than that to synthetic molecules CC (e.g., TLR8 has Kd values of 55 μM TLR9/ssDNA CpG-DNA and 0.2 μM for uridine and R848, respectively). The lower affinity for mononucleosides can be attributed to the lack of the alkyl group harbored by the synthetic ligand, protruding into the hydrophobic pocket of the N N receptor generated by the agonistic CC iDNA form. However, the affinity of TLR7 hydrophobic pocket TLR9/iDNA (b) (c) and TLR8 to mononucleosides could be greatly increased by the Phe405 binding of ssRNA with a shift in TLR8 Kd from 55 μM to 1 μM. This observation was confirmed in a cell- Asp543 based immune response–activation assay showing that uridine stimulated substantial NF-κB activity only in the Fig. 3. Structures of nucleic acid-sensing TLRs presence of a UG-rich ssRNA. The (a) Signaling complexes of nucleic acid-sensing TLRs. Front (left panels) ant top (right panels) views of the signaling synergistic activation is also observed complexes: TLR8/ssRNA (PDB: 4R07), TLR7_G_ssRNA for guanosine derivatives and (PDB: 5GMF), and TLR9/CpG DNA (PDB: 3WPC). (b) Ligand chemical ligands (e.g., loxoribine) in recognition by TLR8. The hydrogen bond is depicted as a red the presence of oligonucleotides in dotted line. (c) Structure of TLR9 in complex with iDNA. TLR7 [3,7]. Likewise, a synergistic activity between uridine analogs and ssRNA is demonstrated in TLR8 containing DNA was recognized demonstrated that CpG methylation [7]. These findings suggest that an by its binding to the groove in the weakens the affinity of CpG to TLR9 oligonucleotide binding at the second N-terminus of TLR9 of one protomer, and its ability to cause receptor site increases receptor affinity to while the C-terminal domain of dimerization, probably because mononucleosides or chemical ligands the other protomer mainly binds of the disruption of water clusters at the first site, possibly via allosteric to the DNA backbone. Thus, CpG- that mediate interactions between regulation. containing DNA acts as the molecular CpG and TLR9. In the TLR9- Accumulating evidence suggests glue bridging two TLR9 monomers. iDNA complex, iDNA, which has a that proteolytic processing in In addition, not only the CpG motif stem-loop conformation stabilized endolysosomes is required for but also its flanking regions are by intramolecular base-pairing, generating functional, mature recognized by TLR9. It is generally demonstrates a close fit to the concave TLRs. This requirement as well accepted that TLR9 distinguishes surface of TLR9 (Fig. 2(c)). Binding as localization within endosomal pathogenic DNA partially on the basis interfaces of CpG-DNA and iDNA compartments reinforced the of methylation status. Our works are partially overlapped. As iDNA prevention of the activation of

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TLRs by self-derived nucleic acid. TLR8, and TLR9 are connected Structural information on the In addition, proteolytic digestion and involved in ligand binding after nucleic acid-sensing TLRs adds of nucleic acid-sensing TLRs may Z-loop cleavage [3,4,6]. Biochemical to and provides new insights restrain inappropriate activation and biophysical studies have revealed into the areas of the regulation in response to host DNA/RNA. that the uncleaved Z-loop prevents of nucleic acid-sensing TLRs, The processing at the Z-loop of the formation of the TLR8 dimer, proteolytic processing of TLR, human TLR8 mediated by furin-like which is essential for its activation synergistic activation mechanism proprotein convertase and cathepsins [10]. Crystallographic analysis through multiple ligand-binding produces functional TLR8 capable demonstrated that the uncleaved sites, and nucleic acid degradation. of ligand binding and signaling in Z-loop located on the ascending In particular, the requirement of endolysosomes. In addition, the lateral face prevents the approach DNA and RNA processing for the cleaved form of TLR8 has been of the dimerization partner by steric activation of immune responses found to be predominant in immune hindrance. Similarly to TLR8, is a potentially paradigm-shifting cells. However, after proteolytic TLR7 and TLR9 also contain the discovery, emphasizing the cleavage, the extracellular and Z-loop, and thus, these proteins importance of specific enzymes, such intracellular domains must remain might also have this autoinhibition as RNases, DNases, and phosphatases associated to provide the functional mechanism. This notion is supported (Fig. 4). Indeed, a recent study shows activity of the receptor [8,9]. This by the observation that TLR9 with that DNA digestion by DNase II is notion is supported by structural the uncleaved Z-loop was unable to required for TLR9 activation [11]. studies demonstrating that the N- dimerize irrespective of CpG-DNA These newly obtained mechanistic and C-terminal domains of TLR7, presence [6]. insights may be a crucial step towards developing therapeutic drugs (agonists and antagonists) that target TLRs for the modulation of host Viruses response to pathogens.

Bacteria Tissue damage Endosome DNaseII RNase? Phosphatase? Toshiyuki Shimizu

O O NH Graduate School of Pharmaceutical N NH

N O N Sciences, The University of Tokyo N NH2 HO ssRNA O HO O

OH OH OH OH ssDNA uridine guanosine Email: [email protected] degradationd products proteasee Z-loop R R RU U L R L References [1] C.A. Janeway et al.: Annu. Rev. Immunol. 20 (2002)197. [2] T. Kawai et al.: Nat. Immunol. 11 (2010) 373. TIR TIR TIR Synthetic [3] Z. Zhang et al.: Immunity 45 (2016) ligands 737. [4] H. Tanji et al.: Science 339 (2013) 1426. [5] H. Tanji et al.: Nat. Struct. Mol. Biol. Fig. 4. Proposed regulation mechanism of nucleic acid-sensing TLRs 22 (2015) 109. Pathogen- and self-derived nucleic acids can be taken up to [6] U. Ohto et al.: Nature 520 (2015) endolysosomes where they are digested by appropriate enzymes 702. (DNase II, RNase, and/or phosphatase) to generate nucleosides and [7] T. Shibata et al.: Int. Immunol. 28 degradation products that are recognized by TLRs. TLRs 7–9 with the (2015) 211. uncleaved Z-loop are monomers. Following Z-loop cleavage, TLRs 7–9 [8] M.M. Hipp et al.: J. Immunol. 194 transforms into the activated dimer upon binding to processed nucleic (2015) 5417. acid agonists. In one example, TLR8 activated by uridine and ssRNA is [9] M. Onji et al.: Nat. Commun. 4 (2013) 1949. shown. Synthetic ligands can directly activate TLR7/8. [10] H. Tanji et al.: Proc. Natl. Acad. Sci USA 113 (2016) 3012. [11] M.P. Chan et al.: Nat. Commun. 6 (2015) 5853.

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