The Structural Biology of Toll-Like Receptors

Structure Review

The Structural Biology of Toll-like Receptors

Istvan Botos,1 David M. Segal,2 and David R. Davies1,* 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases 2Experimental Immunology Branch, National Cancer Institute National Institutes of Health, Bethesda, MD 20892, USA *Correspondence: [email protected] DOI 10.1016/j.str.2011.02.004

The membrane-bound Toll-like receptors (TLRs) trigger innate immune responses after recognition of a wide variety of pathogen-derived compounds. Despite the wide range of ligands recognized by TLRs, the receptors share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, on ligand binding, two extracellular domains form an ‘‘m’’-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signaling cascade. Although the ligand-induced dimerization of these receptors has many common features, the nature of the interactions of the TLR extracellular domains with their ligands varies markedly between TLR paralogs.

Introduction proteins that interact homotypically with the TIR domains of In the initial phase of an infection, the innate immune system TLRs and IL-1 receptors as the first step in the signaling generates a rapid inflammatory response that blocks the growth cascade. Remarkably, homologs of TIR domains are also found and dissemination of the infectious agent. This response is in some plant proteins that confer resistance to pathogens followed, in vertebrates, by the development of an acquired (Burch-Smith and Dinesh-Kumar, 2007), suggesting that the immune response in which highly specific B and T cell receptors TIR domain represents a very ancient motif that served an recognize the pathogen and induce responses that lead to its immune function before the divergence of plants and animals. elimination (Janeway and Medzhitov, 2002). The antigen recep- The transmembrane domains of TLRs each contain a typical tors of the acquired immune system are well characterized. They stretch of 20 uncharged, mostly hydrophobic residues. TLRs consist of many structurally similar molecules with different that recognize nucleic acid PAMPs interact through their trans- binding specificities created by somatic rearrangements and membrane domains with a multispan transmembrane protein mutations within the binding site regions of the B and T cell known as UNC93B, which directs these TLRs to endocytic receptor variable domains (Jung and Alt, 2004; Schatz and compartments (Brinkmann et al., 2007; Kim et al., 2008). The re- Spanopoulou, 2005). By contrast, the receptors of the innate maining TLR paralogs do not interact with UNC93B, and traffic immune system are germline-encoded and have been selected directly to the cell surface. The N-terminal ectodomains (ECDs) by evolution to recognize pathogen-derived compounds that of TLRs are glycoproteins with 550–800 amino acid residues are essential for pathogen survival or endogenous molecules (Bell et al., 2003). These ectodomains are either extracellular or that are released by the host in response to infection (Matzinger, in endosomes where they encounter and recognize molecules 1994; Yang et al., 2010; Erridge, 2010). Innate immune receptors, released by invading pathogens. We review our current under- also known as pattern recognition receptors (PRRs), have been standing of the structural basis for ligand recognition and signal identified in the serum, on the cell surface, in endosomes, and transduction by TLRs. in the cytoplasm (Medzhitov, 2007). The Toll-like receptors (TLRs) represent a particularly important group of PRRs Leucine-Rich Repeats—The Building Blocks of TLRs (Gay and Gangloff, 2007). TLR paralogs are located on cell All TLR ECDs are constructed of tandem copies of a motif known surfaces or within endosomes and have important roles in the as the leucine-rich repeat (LRR), which is typically 22–29 resi- host defense against pathogenic organisms throughout the dues in length and contains hydrophobic residues spaced at animal kingdom. In humans, 10 TLRs respond to a variety of distinctive intervals (Figure 1A). This motif is found in many pathogen-associated molecular patterns (PAMPs), including proteins in animals, plants and microorganisms, including lipopolysaccharide (TLR4), lipopeptides (TLR2 associated with many proteins involved in immune recognition (Palsson-McDer- TLR1 or TLR6), bacterial flagellin (TLR5), viral dsRNA (TLR3), viral mott and O’Neill, 2007). In three dimensions, all LRRs adopt or bacterial ssRNA (TLRs 7 and 8), and CpG-rich unmethylated a loop structure, beginning with an extended stretch that DNA (TLR9), among others (Kumar et al., 2009). contains three residues in the b strand configuration that were The TLRs are type I integral membrane receptors, each with an recently reviewed (Bella et al., 2008)(Figure 1B). When assem- N-terminal ligand recognition domain, a single transmembrane bled into a protein, multiple consecutive LRRs form a solenoid helix, and a C-terminal cytoplasmic signaling domain (Bell structure, in which the consensus hydrophobic residues point et al., 2003). The signaling domains of TLRs are known as Toll to the interior to form a stable core and the b strands align to IL-1 receptor (TIR) domains because they share homology with form a hydrogen-bonded parallel b sheet. Because the b strands the signaling domains of IL-1R family members (O’Neill and are more closely packed than the non-b portions of the LRR Bowie, 2007). TIR domains are also found in many adaptor loops, the solenoid is forced into a curved configuration in which

Figure 1. The Structure of Leucine-Rich Repeats (A) LRR consensus sequences for TLR3 and ribonuclease inhibitor. Residues forming the b strand are highlighted in orange. (B) A LRR loop from hTLR3 and a LRR loop from RI, with the conserved residues forming a hydrophobic core. The boxed regions form the surfaces involved in ligand binding. (C) Ribbon diagram of TLR3 (Bell et al., 2005) (2A0Z) and ribonuclease inhibitor (Kobe and Deisenhofer, 1995) (1DFJ). (D) Ribonuclease inhibitor complexed with ribonuclease A (Kobe and Deisenhofer, 1995) (1DFJ). The LRR protein is shown in blue and the ligand in orange. (E) Lamprey VLR complexed with H-trisaccharide (Han et al., 2008) (3E6J). (F) Glycoprotein Ib a complexed with the von Willebrand factor A1 domain (Huizinga et al., 2002) (1M10). Figures generated with program Pymol (DeLano, 2002).

and a descending lateral surface on the opposite side (Bella et al., 2008). The ﬁrst LRR protein structure described was ribonuclease inhibitor (RI) (Kobe and Deisenhofer, 1995). The LRRs in this protein are relatively long, typically 27–29 amino acids in length, and all LRRs have three to four turns of a-helix on their convex surfaces opposite the b sheet. RI contains 16 LRRs that do not form a complete circle, but form a ‘‘horseshoe’’ structure (Figure 1C). In the innate immune system, a family of cytoplasmic danger sensors known as ‘‘Nod-like receptors’’ (NLRs) contain nine or fewer contiguous RI-like LRRs at their C-terminal ends, which are thought to be involved in recognizing danger signals. Based on the RI structure, one would predict that the LRRs of the NLR proteins form banana-shaped structures with a b sheet on their concave surfaces and a helices on their convex surfaces. The TLR-ECDs typically contain 19–25 LRRs that, like RI, form horseshoe structures. In contrast to RI, the consensus LRR of the TLRs is 24 residues in length (Figure 1A), which does not allow for the formation of multi-turn helices on their convex sides. Consequently, interstrand distances on the convex side are shorter for TLRs than for RI, which gives rise to TLR-ECD structures with lower curvature and larger outer diameters (90 A˚ ) than for RI (Figure 1C). The 24-residue the concave surface is formed by the b sheet (Kajava, 1998)(Fig- consensus LRRs adopt a variety of conﬁgurations on their ure 1C). As a result, each LRR protein contains a concave convex sides, often containing bits of secondary structure surface, a convex surface, an ascending lateral surface that such as b strands, 310 helices, and polyproline II helices. In other consists of loops connecting the b strand to the convex surface, proteins that contain 24-residue LRRs, for example glycoprotein

Table 1. Main Features of the 10 Human TLR Molecules N-Linked TLR Residues LRRsa Glycosylation Sitesb Accession Code 1 786 19 4 (7) Q15399 2 784 19 3 (4) O60603 3 904 23 11 (15) O15455 4 839 21 5 (10) O00206 5 858 20 (7) O60602 6 796 19 8 (9) Q9Y2C9 7 1049 25 (14) Q9NYK1 8 1041 25 (18) Q9NR97 9 1032 25 (18) Q9NR96 10 811 19 (8) Q9BXR5 LRR, leucine-rich repeat; TLR, Toll-like receptors. a The number of LRRs in the extracellular domain do not include the LRR-NT or LRR-CT motifs. b Number of N-glycosylation sites observed in the crystal structure or predicted by the NetNGlyc server 1.0 in parentheses (http://www.cbs. dtu.dk/services/NetNGlyc/).

generate great diversity by randomly joining LRRs from a large pool of genomically encoded LRR cassettes into the mature VLR protein. In these molecules, the highest diversity in amino acid sequence occurs on the concave b surface. In the two ligand-VLR structures available, binding occurs on this surface (Deng et al., 2010; Han et al., 2008; Velikovsky et al., 2009) (Figure 1E). In addition, a large loop that interacts with the ligand protrudes from the LRR-CT in both structures, which is also seen Figure 2. The Structure of a TLR-ECD (hTLR3) in the glycoprotein Iba-von Willebrand factor complex (Huizinga Top and side views of the TLR3-ECD, with the N-linked glycosyl moieties et al., 2002)(Figure 1F). By contrast, in the known TLR-ligand (Bell et al., 2005) (2A0Z). The LRRs are capped by the LRR-NT and LRR-CT motifs and the molecule is flat with one glycan-free side (ascending side) that is structures, ligand binding occurs most often on the ascending involved in receptor dimerization. lateral surface of the TLR-ECD (Jin et al., 2007; Kang et al., 2009; Liu et al., 2008; Park et al., 2009)(Figure 2). This surface is the only portion of the molecule that completely lacks N-linked Iba (Uff et al., 2002), Nogo receptor (He et al., 2003), and the vari- glycan and is free to interact with a ligand. able lymphocyte receptors (VLRs) of jawless vertebrates among others, a slight twist relates each LRR to its neighbor, which generates a nonplanar overall structure. The twist in these The Structure of TLRs proteins is most likely due to the inherent twist found in b sheets. Based on sequence homologies, vertebrate TLRs can be By contrast, the known TLR-ECD structures are more planar, grouped into six subfamilies, TLR1/2/6/10, TLR3, TLR4, TLR5, suggesting that structures on the convex and lateral sides of TLR7/8/9, and TLR11/12/13/21/22/23 (Matsushima et al., TLR LRRs counteract the twist tendency on the concave side. 2007; Roach et al., 2005). Not all vertebrate species express all Planarity of TLR-ECDs may be important for ligand binding and TLR paralogs; humans, for example lack all members of activation (see below). the TLR11 family. As indicated in Table 1, the ECDs of the A characteristic feature of TLR-ECDs is the frequent occurrence 10 human TLRs vary in LRR numbers and extent of N-linked of LRRs that are substantially larger than the consensus 24 resi- glycosylation. To date, the structures of the ECDs of TLRs 1, 2, dues, especially in TLRs 7, 8, and 9. These extra residues often 3, 4, and 6 (human or mouse) have been reported (Table 2). All produce loops that protrude from the TLR-ECD horseshoe, usually ECDs assume the typical horseshoe-shape, but their structures on the ascending or convex side of the LRR (Figure 1B). The cannot be superimposed because of variations in curvature. TLR-ECDs also contain structures that cap the N and C-terminal In the known structures, the glycans are distributed throughout ends known as the LRR-NT and LRR-CT motifs, respectively the molecule, except for the lateral face formed by the ascending (Figure 2). The LRR-NTs are disulfide-linked b-hairpins, whereas loops of the LRRs (Figure 2). This glycan-free face is involved in the LRR-CTs are globular structures that contain two a helices dimerization on ligand binding in the known TLR/ligand struc- and are stabilized by two disulfide bonds. Similar capping motifs tures (see below). have been observed in several other proteins that contain 24-residue LRRs (He et al., 2003; Huizinga et al., 2002). The TLR1/2/6/10 Subfamily In most LRR proteins, ligand binding occurs on the concave TLR2 resides on the plasma membrane where it responds to surface (Figure 1D). For example, the VLRs of jawless vertebrates lipid-containing PAMPs such as lipoteichoic acid and di- and

Table 2. TLR-ECD and TIR domain structures Residues Molecule TLR\VLR Resolution A˚ PDB Code Reference TLR monomers a hTLR2 T1–284\V136–199 1.8 2Z80 Jin et al., 2007

mTLR2/Pam3CSK4 T27–506\V136–199 1.8 2Z81 Jin et al., 2007

mTLR2/Pam2CSK4 T27–506\V133–199 2.6 2Z82 Jin et al., 2007

mTLR2/pnLTA T27–506\V133–200 2.5 3A7B Kang et al., 2009

mTLR2/PE-DTPA T27–506\V133–200 2.4 3A7C Kang et al., 2009

hTLR3 T22–696 2.4 2A0Z Bell et al., 2005

hTLR3 T27–664 2.1 1ZIW Choe et al., 2005

mTLR3 T27–697 2.6 3CIG Liu et al., 2008

hTLR4 T27–228\V128–199 1.7 2Z62 Kim et al., 2007

hTLR4 T27–527\V133–199 2.0 2Z63 Kim et al., 2007

hTLR4 V24–82\T228–383 1.9 2Z66 Kim et al., 2007

hTLR4/hMD-2/Eritoran T27–228\V19–158 2.7 2Z65 Kim et al., 2007

mTLR4/mMD-2 T27–625 2.8 2Z64 Kim et al., 2007 hMD-2 2.0 2E56 Ohto et al., 2007 hMD-2/lipid IVa 2.2 2E59 Ohto et al., 2007 TLR dimeric complexes

hTLR1/hTLR2/Pam3CSK4 T25–475\V133–199, 2.1 2Z7X Jin et al., 2007

T27–506\V133–199

mTLR3/dsRNA T28–697 3.4 3CIY Liu et al., 2008

hTLR4/MD-2/LPS T27–631 3.1 3FXI Park et al., 2009

mTLR2/mTLR6/Pam2CSK4 T26–506\V133–200, 2.9 3A79 Kang et al., 2009

T33–482\V157–232 TIR domains hTIR1a 2.9 1FYV Xu et al., 2000 hTIR2 3.0 1FYW Xu et al., 2000 hTIR2 P681H mutant 2.8 1FYX Xu et al., 2000 hTIR2 C713S mutant 3.2 1O77 Tao et al., 2002 hTIR10 2.2 2J67 Nyman et al., 2008 TIR (human IL-1RAPL) 2.3 1T3G Khan et al., 2004 TIR (Arabidopsis) 2.0 3JRN Chan et al., 2010 pdTIR (Paracoccus) 2.5 3H16 Chan et al., 2009 TIR (MyD88) NMR 2Z5V Ohnishi et al., 2009 TIR (MyD88) NMR 2JS7 Unpublished NMR, nuclear magnetic resonance; PDB, Protein Data Bank; PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriamine- pentaacetic acid (synthetic phospholipid derivative); pnLTA, Streptococcus pneumoniae lipoteichoic acid; TIR, Toll IL-1 receptor; TLR, Toll-like receptors; VLR, variable lymphocyte receptors. Available in the PDB as of 2010. a Human (h) and mouse (m) proteins with the residue numbers shown for the TLR part (T) and the VLR part (V) in case of the chimeric constructs. The length of the full TLR-ECDs including the signal peptide: TLR1 577 residues, TLR2 585, TLR3 696, TLR4 627, and TLR6 582.

tri-acylated cysteine-containing lipopeptides (Takeda et al., C-terminal (Figure 3A). The N-terminal subdomain contains the 2003). It does this by forming dimeric complexes with either LRR-NT capping motif and LRRs 1–4, with typical 24-residue TLR1 or TLR6 on the plasma membrane. The structures of LRR modules. In this region, two features commonly found in TLR2/TLR1 and TLR2/TLR6 with lipopeptide ligands have been the interiors of LRR structures are preserved: an asparagine determined (Jin et al., 2007; Kang et al., 2009). To facilitate crys- ladder, formed by a continuous network of hydrogen bonds tallization and structure determination the LRR-CT and the last between consensus asparagines (Figure 1A) and neighboring one or two LRRs of TLRs 1, 2, and 6 were replaced by corre- backbone oxygens (Kajava, 1998); and a spine formed by sponding regions of a hagﬁsh VLR (Table 2)(Kim et al., 2007). consecutive consensus phenylalanine residues (Figure 1A) (Jin Structural analyses revealed that the ECDs of TLRs 1, 2, and 6 et al., 2007). By contrast, the central and C-terminal subdomains contain three distinctive subdomains: N-terminal, central, and have LRR modules with atypical sequences, and their b sheet

Figure 3. Structure of the TLR1/TLR2/ Pam3CSK4 Complex (A) The TLR2-ECD showing the position of the three subdomains: N-terminal, central, and C-terminal. (B) hTLR1 and Pam3CSK4 ligand interactions mapped on the molecular surface. (C) hTLR2 and Pam3CSK4 interactions mapped on the surface. (D) Ribbon diagram of TLR1/TLR2 binding Pam3CSK4 (magenta) (Jin et al., 2007) (2Z7X).

the dimerization of the TIR domains on the cytoplasmic side of the plasma membrane. Whereas the TLR1/2 complex recognizes tri-acylated lipopeptides such as

Pam3CSK4, the TLR2/6 complex recognizes the di-acylated ligand, Pam2CSK4. The latter ligand lacks the lipid chain that binds in the hydrophobic pocket of TLR1, raising the question of how the diacylated lipopeptide is able to stabilize a complex between TLRs 2 and 6. The crystal structure of the TLR2/TLR6/

Pam2CSK4 complex (Kang et al., 2009) indicates that TLR6 and TLR1 have very conformations deviate from those seen in standard LRRs. The similar, horseshoe structures and that the TLR2/6 complex central subdomain is also lacking the asparagine ladder and adopts the same overall m-shaped structure as seen in the phenylalanine spine. TLR1/2 complex (Figure 4B). However, TLRs 1 and 6 contain The border between the central and C-terminal subdomains important structural differences in their ligand binding and dimer- (LRRs 9–12) harbors ligand-binding pockets on the convex ization regions. In TLR6, the side chains of two phenylalanine side in both TLR1 and TLR2 ECDs (Figures 3B and 3C). These residues block the lipid binding channel, leading to a channel pockets are lined with hydrophobic residues and can accommo- that is less than half the length as that of TLR1 (Figure 4C). date fairly long lipid chains from lipopeptide ligands. The shapes This structural feature provides selectivity for diacylated over of lipid binding pockets vary between species (Jin et al., 2007). triacylated lipopeptides, as confirmed by the observation that For example, the mouse TLR2 binding pocket is shorter than mutation of the F343 and F365 residues in TLR6 to their TLR1 the human. As a result, mouse TLR2 binds relatively short lauryl counterparts allows TLR6 to respond to the triacylated chains more efficiently than human TLR2, which is also reflected Pam3CSK4 (Kang et al., 2009). In the TLR2/6 complex, the two in the capacity of lauryl3CSK4 to activate mouse, but not human lipid chains of Pam2CSK4 are buried in the TLR2 hydrophobic TLR2. In the TLR1/TLR2 complex (Figure 3D), the triacylated pocket as in the TLR1/2 complex whereas the peptide part of lipopeptide ligand, Pam3CSK4, bridges the two TLR-ECDs by Pam2CSK4 forms several hydrogen bonds with both TLR2 and inserting the two ester-bound palmitoyl groups into the TLR2 TLR6 (Figure 4C). The LRR11 loop in TLR6 is slightly displaced binding pocket, and the single amide-bound palmitoyl chain relative to its position in TLR1 and forms a prominent hydrogen into the TLR1 pocket (Jin et al., 2007) (Figures 3B and 4A). bond with the carbonyl oxygen of the first peptide bond of the

TLRs 1 and 2 also interact with the head group of Pam3CSK4 ligand. In addition, the LRR11-LRR14 regions of TLR2 and by forming hydrogen bonds with the glycerol and peptide TLR6 form a heterodimeric interface via hydrophobic and hydro- portions and by forming hydrophobic interactions with the sulfur philic interactions of their surface-exposed residues. The area of atom. Importantly, TLRs 1 and 2 interact with each other on their hydrophobic interaction is 80% larger than in the hTLR1/hTLR2 dimerization surfaces via several hydrogen bonds and hydro- complex, suggesting that this surface interaction together with phobic interactions in the vicinity of the binding pockets. The the hydrogen bond between LRR11 and the ligand drives the importance of these interactions to TLR1/2 function is suggested heterodimerization of TLR6. by the observation that a polymorphic mutation in this region, Lipoteichoic acid (LTA), a glycolipid from Gram-positive P315L, abolishes its ability to respond to lipopeptide ligands bacterial membranes contains a diacylated glycerol group con- (Omueti et al., 2007). The ligand-protein and protein-protein nected by an ether bond to a variable carbohydrate moiety. interactions bring the C-terminal regions of the ECDs in close LTAs vary in their capacity to activate TLR2, for example Staph- proximity, giving rise to an overall structure of the complex that ylococcus aureus LTA is a much more potent activator than has been described as having an ‘‘m’’-shape (Figure 3D). The Streptococcus pneumoniae LTA. The crystal structure for close apposition of the C-terminal regions likely facilitates S. pneumoniae LTA bound to the TLR2/VLR hybrid shows

Figure 4. Ligand Binding by TLR 1, 2, and 6 (A) Cross-section through the molecular surface of the TLR1/TLR2/Pam3CSK4 complex (2Z7X). (B) Ribbon diagram of TLR2/TLR6 binding Pam2CSK4 (magenta) (Kang et al., 2009) (3A79). (C) Cross-section through the molecular surface of the TLR2/TLR6/Pam2CSK4 complex (3A79). (D) Cross-section through the molecular surface of the TLR2/pnLTA complex (Kang et al., 2009) (3A7B). (E) Cross-section through the molecular surface of the TLR2/PE-DTPA complex (Kang et al., 2009) (3A7C).

phobic interactions with the TLR1 channel, and hydrophobic interaction of the conserved cysteine from peptide headgroups. The heterodimer is further stabilized by hydrophobic, hydrogen- bonding, and ionic interactions between the TLR molecules. Phylogenic analysis identifies TLR10 as part of the TLR-1 family (Roach et al., 2005). Based on the above TLR1/TLR2 and TLR2/TLR6 complex structures, homology models of hTLR10 complexes were constructed and refined through molecular dynamics simulations (Govin- daraj et al., 2010). The hTLR2/hTLR10 and hTLR1/hTLR10 complex models were similar to the available TLR1 family complexes. However, the binding orientation of the hTLR10 homodimer was different due to the presence of nega- tively charged surfaces near LRR 11–14 that define a specific binding pocket. Docking experiments suggest that

Pam3CSK4 might be a ligand for the differences compared to lipopeptide bound to TLR2 (Kang et al., hTLR2/hTLR10 complex and PamCysPamSK4 might bind to 2009)(Figure 4D). Curiously, this LTA failed to dimerize TLR2 or hTLR1/hTLR10 and the hTLR10 homodimer. induce hetero-dimers with TLR1 or TLR6, although it was capable of activating TLR2 in cells, probably by forming TLR3 a complex with TLR1 (Han et al., 2003). Another nonpeptidic TLR3 recognizes dsRNA, which is produced by most viruses at ligand PE-DTPA (a synthetic derivative of phosphatidylethanol- some stage in their life cycles and is a potent indicator of viral amine) binds mTLR2/VLR similar to LTA (Kang et al., 2009) infection. In contrast to several cytoplasmic receptors for (Figure 4E). These TLR2/ligand structures are missing the dsRNA, TLR3 is localized to endosomes where it recognizes hydrogen bonding network present in TLR2-lipopeptide dsRNA. Studies using soluble TLR3-ECD protein (Leonard complexes, due to the shift of the carbohydrate head groups et al., 2008) or immobilized TLR3-GFP constructs (Wang et al., relative to the peptide head groups of lipopeptide ligands. This 2010) showed that TLR3-ECD is monomeric in solution but binds shift is also enhanced by the repulsion between the head group as dimers to 45-bp segments of dsRNA, the minimum length oxygen atom of the ligand and the hydrophobic sulfur binding required for TLR3 binding and activation. In addition, binding is site of TLR2, which is normally filled by the sulfur atom from independent of base sequence and occurs only at pH 6.5 and the lipopeptide cysteine. The hydrogen bonding network is below (Leonard et al., 2008). A crystal structure of the TLR3- possibly required to maintain the correct conformation of the ECD dimer complexed with a 46-bp dsRNA oligonucleotide TLR2 LRR11 loop that makes crucial intermolecular contacts explains these properties. with TLR1 or TLR6 in the heterodimeric complexes. The first reported crystal structure of a TLR binding domain There are four different types of protein-ligand interactions for was that of the unliganded form of TLR3-ECD (Bell et al., 2005; the TLR 1, 2, and 6 complexes. These interactions can be ranked Choe et al., 2005)(Figure 2). The TLR3-ECD horseshoe is largely in order of their importance: hydrophobic interactions with the uniform and flat, lacking the subdomain structure seen in TLRs 1, TLR2 pocket, hydrogen bonding of peptide head groups, hydro- 2, 4, and 6. However, irregularities in the LRRs produce short

et al., 2008). Because cells normally contain short (%25 bp) stretches of dsRNA for example in miRNA and tRNA hairpins, the inability of TLR3 to bind dsRNA <40 bp most likely provides an important mechanism for preventing autoreactive responses against self dsRNA. In binding dsRNA, the TLR3-ECD interacts only with the ribose-phosphate backbone, accounting for the lack of RNA sequence speciﬁcity in binding. This feature would prevent the viruses from escaping detection by mutation. All binding-site residues are located on the glycan-free surface of the ECD. Of a particular interest is the Asn413 N-linked pauci- manose moiety that reaches out from the concave surface and contacts the dsRNA helix, but the relevance of this interaction is unclear. The majority of TLR3/dsRNA interactions are hydrophilic, involve hydrogen bonds and salt bridges, and account for a total buried area of 1103 A˚ 2 (4.4% of the TLR3-ECD surface). Partic- ularly important are electrostatic interactions between the phosphate groups from the dsRNA backbone and the imidazole rings of four histidine residues, three in the N-terminal site, and one in the C-terminal site. Mutation of two of these histidines (H39 and H60 in the N-terminal site) to alanine abolishes binding and responsiveness to dsRNA, indicating that the salt bridges formed by these residues are essential for complex formation. This could explain the pH-dependency of dsRNA binding because these side chains would be protonated only below pH 6.5 and able to interact with the RNA phosphates. The structure of the complex reveals the main reasons for the inability of TLR3 to interact with dsDNA. The helical structure of dsDNA is the B form, whereas dsRNA assumes the A form. The B helix Figure 5. Structure of the TLR3/dsRNA Complex would not be structurally compatible with the two terminal (A) Molecular surface of TLR3 dimer (green) with bound dsRNA (Liu et al., 2008) (3CIY). The interaction of the C-terminal capping motifs stabilizes the TLR3 binding sites on the TLR3-ECD horseshoe. Also, there are 0 dimer. several hydrogen bonds between TLR3-ECD and the 2 -hydroxyl (B) Top view. groups of dsRNA that would be missing in dsDNA. The only interaction between the two TLR3 molecules in the complex occurs at the interface between the two LRR-CT motifs, alpha helices on the convex side of the horseshoe and two large, and therefore this site is responsible for dimerization. In this site, conserved loops that protrude from the lateral and convex faces the two LRR-CTs are related by two-fold symmetry and the inter- of LRRs 12 and 20, respectively. TLR3-ECD is heavily glycosy- actions between the LRR-CTs are mainly hydrophilic, consisting lated, with 15 predicted N-glycosylation sites, of which 11 are of hydrogen bonds and salt bridges. The dimerization site is visible in the crystal structure. The glycans decorate all faces essential for dsRNA binding (Wang et al., 2010) because it of the TLR3-ECD, except for the lateral surface on the C-terminal correctly positions the four dsRNA binding sites in the complex, side of the b sheet. This face offers a large, planar surface for but in addition it also brings the two C-terminal residues within interaction with dsRNA (Figure 2). In the crystal structure of the 25 A˚ of each other. In the cell, two LRR-CT motifs that interact TLR3-ECD-dsRNA complex, the glycan-free surfaces of two on the luminal side of an endosome would presumably bring the TLR3-ECDs sandwich the dsRNA molecule, generating an two TIR domains together on the cytoplasmic side, forming m-shaped structure (Liu et al., 2008)(Figure 5A). No conforma- a dimeric scaffold on which adaptor molecules could bind and tional change in the TLR3-ECD occurs on ligand binding. In the initiate signaling. complex, the dsRNA interacts at two sites on each TLR3-ECD, one near the N terminus (encompassing LRR-NT and LRRs 1-3), and one near the C terminus (involving LRRs 19–23) TLR4 and MD-2 (Figure 5B). In addition, the two ECDs interact with each other Lipopolysaccharide (LPS), an essential component of the outer at their LRR-CT motifs. Mutational analyses (Wang et al., 2010) membrane of Gram-negative bacteria, induces a powerful have established that the simultaneous interaction of all three inﬂammatory response that can lead to septic shock and death sites is required for stable binding of dsRNA to TLR3. (Beutler and Rietschel, 2003). LPS signals through TLR4 by com- Although the N- and C-terminal dsRNA sites are separated by plexing coreceptor MD-2, which is anchored by several 55–60 A˚ in each ECD, the two N-terminal sites in the complex are hydrogen bonds to the lateral and concave surface of TLR4- separated by 110 A˚ . This latter distance correlates with a dsRNA ECD and contacts residues from the LRR2-LRR10 area length of 45 base pairs and explains why dsRNA oligonucleo- (Kim et al., 2007)(Figure 6A). LPS-binding protein (LBP) and tides less than 40 bp cannot bind or activate TLR3 (Leonard CD14 deliver and load the LPS to the TLR4-bound MD-2.

Figure 6. Structure of the TLR4/MD-2/LPS Complex (A) Ribbon diagram of TLR4 (green), MD-2 (blue) binding LPS (magenta) (Park et al., 2009) (3FXI). (B) Cross-section through the molecular surface of the MD-2/LPS complex. (C) TLR4 and LPS interactions mapped on their molecular surfaces. (D) Cross-section through the molecular surface of the MD-2/lipid IVa complex (Ohto et al., 2007) (2E59). (E) Cross-section through the molecular surface of the MD-2/Eritoran complex (Kim et al., 2007) (2Z65).

fragments of TLR4 capped with hagfish VLR domains (Kim et al., 2007) show a horseshoe structure that is much less planar than the TLR3-ECD. Three subdomains can be differentiated, N-terminal, central, and C-terminal, with different degrees of twist and curvature. The central subdomain contains only one variable residue between the last leucine residue of a preceding LRR motif and the first leucine residue of the next LRR motif, in contrast with the standard two variable residues (Figure 1A). The length of these LRR modules also varies between 20–30 residues, conferring a smaller radius and greater twist angle to the subdomain. The central subdomains of human and mouse TLR4 differ most in structure, apparently due to the MD-2 binding to the mTLR4 LRR9 loop. MD-2 is a coreceptor molecule that binds both the extracellular domain of TLR4 and the hydrophobic portion of LPS (Visintin et al., 2006). The crystal structure of hMD-2 complexed to lipid IVa has been determined (Ohto et al., 2007)(Figure 6D). Lipid IVa, a precursor One of the important factors that determine the inflammatory of Lipid A, is an antagonist for hTLR4 but an agonist for mTLR4 potential of an LPS molecule (Rietschel et al., 1994) is the (Means et al., 2000). The molecule adopts the b cup topology number of lipid chains in the Lipid A portion of LPS. Six chains of the ML family of lipid-binding proteins that have sandwiched provide an optimal inflammatory activity, whereas Lipid A with antiparallel b sheets and conserved disulfide bridges (Dere- five chains has 100-fold less activity. Ligands with only four wenda et al., 2002). The b sheets of hMD-2 form a very deep lipid chains, such as lipid IVa and Eritoran, have antagonistic and narrow hydrophobic pocket, with a surface area of 1000 activity (Teghanemt et al., 2005). Because the lipids interact A˚ that can bury the four lipid strands of lipid IVa. The opening with the MD-2 pocket through hydrophobic contacts, variations of the pocket is lined by positively charged residues and three in the lipid chain structure can be accommodated by shifting the disulfide bridges stabilize the cup-like structure. Based on this position of the chains in the pocket. With a smaller number of structure, it seemed unlikely that the hMD-2 pocket would be lipid chains, Lipid A can move deeper into the pocket and the able to accommodate more than four lipid chains without confor- phosphate groups cannot participate in ionic interactions with mational changes, but another structure with bound LPS TLRs (Park et al., 2009). These two phosphate groups are also showed that additional room for the ligand is generated, not by essential for the endotoxic activity of LPS, and deleting either conformational change, but by displacing the LPS glucosamine of them reduces the endotoxic activity by 100-fold (Rietschel backbone upward by 5A˚ (Park et al., 2009). In addition to the et al., 1994). displacement, the glucosamine backbones are also rotated by TLR4-ECD has 21 LRRs capped by LRR-NT and LRR-CT 180, interchanging the two phosphate groups. The crystal motifs. The structures of the TLR4-ECD and several chimeric structure of the TLR4-ECD/MD-2/Eritoran complex was also

reported (Kim et al., 2007)(Figure 6E). Eritoran is a synthetic 2003). Thus it is likely that LRR9 plays an important role in antagonist with four lipid chains that occupy 90% of the flagellin recognition by TLR5. hMD-2 hydrophobic pocket, whereas the di-glucosamine sugars are fully exposed to solvent and the phosphate groups form The TLR 7, 8, and 9 family essential ionic bonds with positively charged residues at the No structure has yet been reported for any member of the TLR 7, opening of the pocket. Binding of Eritoran does not induce signif- 8, and 9 subfamily. Like TLR3, these TLRs are located in endo- icant structural changes in hMD-2. The available structures somes and recognize nucleic acid PAMPs. However, their amino suggest that the MD-2 pocket evolved to bind large and structur- acid sequences suggest that the structures of TLRs 7-9 ECDs ally different ligands (Kim et al., 2007). Agonists and antagonists are markedly different from TLR3 (Bell et al., 2003). TLRs 7–9 will bind in a similar fashion, but with their glucosamine back- ECDs each comprise 25 LRRs and are heavily glycosylated. bones rotated by 180. They all contain large insertions in LRRs 2, 5, and 8 that most The 3.1 A˚ resolution structure of the TLR4/MD-2/LPS complex likely give rise to structures that loop out from the dimerization (Park et al., 2009) shows two Escherichia coli LPS molecules surfaces of the ECDs. In addition, the ECDs of TLRs 7–9 contain bound (Figure 6A). Five of the LPS lipid chains are buried in the stretches of 40 residues between LRRs 14 and 15 that have large hydrophobic MD-2 pocket, whereas the sixth one is undefined structure. Because these stretches are the only exposed and makes hydrophobic contacts with conserved portions that show a high degree of species variability in each phenylalanines on the other TLR4-ECD (Figure 6B). The LPS of the three paralogs, it is likely that these regions are relatively phosphate groups form ionic interactions with positively charged unstructured. TLR9, which recognizes single stranded DNA residues on MD-2 and TLR4-ECD (Figure 6C). In addition, the with unmethylated CpG sequences, especially in viral and bacte- F126 and L87 loops of MD-2 interact with the second TLR4- rial DNA, has been the most extensively studied paralog of this ECD molecule. All these interactions bring the two copies of family (Hemmi et al., 2000; Kumar et al., 2009). There is evidence the TLR4-ECD/MD-2/LPS complex together into a typical (Latz et al., 2007) that TLR9 undergoes an extensive conforma- m-shaped signaling complex. tional change when binding CpG DNA, which would make it Cells that express TLR4/MD-2 also express a homologous very different from TLR3. A ligand-induced conformational complex, RP105/MD-1, that regulates the LPS response change could be dependent on the unstructured region if it (Akashi-Takamura and Miyake, 2008; Divanovic et al., 2007). served as a hinge within the horseshoe, thus allowing a large RP105 is a type I receptor with an ECD resembling that of conformational transition to occur on ligand binding. More TLR4. However, RP105 lacks a C-terminal TIR domain. MD-1, recently, several studies have suggested that proteases are like MD-2, binds LPS and a crystal structure of a lipid IVa/ required for TLR9 function (Asagiri et al., 2008; Ewald et al., MD-1 complex has been reported (Yoon et al., 2010). Although 2008; Matsumoto et al., 2008; Park et al., 2008), and that TLR9 both MD-1 and MD-2 are members of the ML family of lipid is cleaved within the undefined region between LRRs 14 and binding proteins, they use different surfaces for ligand recogni- 15 (Park et al., 2008), consistent with this being an unstructured, tion. RP105/MD-1 binds directly to TLR4/MD-2, and on B cells exposed stretch of amino acid residues. Another report suggests RP105/MD-1 enhances the LPS response (Miyake et al., 1994, that the C-terminal fragment, lacking the first 14 LRRs and the 1998; Miura et al., 1998), whereas on macrophages it attenuates undefined region, is active by itself implying that the N-terminal the response (Divanovic et al., 2005). fragment is not needed for ligand recognition (Park et al., 2008). This observation seems to be inconsistent with a more TLR5 recent study (Peter et al., 2009) showing that mutations within TLR5 is one of the few TLRs that recognize a protein PAMP, the N-terminal fragment inactivate TLR9. Indeed it seems bacterial flagellin (Hayashi et al., 2001). It is highly expressed in unlikely that a large portion of the TLR9-ECD would be highly gut, especially in lamina propria dendritic cells (Uematsu and conserved throughout vertebrate evolution, and yet not be Akira, 2009) where it controls the composition of the microbiota essential for TLR9 function. Clearly, a detailed structural analysis (Vijay-Kumar et al., 2010). With no structure available, TLR5-ECD of the TLR9-ECD is needed to clarify the mechanism of ligand is predicted to contain 20 LRRs, with five loops extending from binding and activation of TLR9 as well as TLRs 7 and 8. the ascending or convex surfaces (Bell et al., 2003). Mutational analyses of flagellin have located the site recognized by TLR5 Common Features and Differences between as lying in the conserved D1 domain (Donnelly and Steiner, the Signaling Complexes 2002). This domain is exposed in monomeric flagellin, but is The signaling complex structures of the TLRs with their respec- buried in the polymerized flagellin fiber, suggesting that TLR5 tive ligands reveal the diverse mechanisms of recognition of recognizes flagellin monomers that are released on depolymer- a wide variety of PAMPs. These PAMPs carry characteristic ization of flagellin polymers (Smith et al., 2003). The portion of molecular signatures that are unique to different classes of path- the TLR5-ECD that interacts with flagellin is less well defined. ogens and are detected by the TLRs. In all structures, the Human and mouse TLR5 differ in their ability to recognize WT signaling complex consists of an m-shaped TLR dimer, in which or mutated flagellin molecules from different sources (Ander- the ECD N-termini extend to the opposite ends and the C-termini sen-Nissen et al., 2007; Miao et al., 2007). Moreover, mouse interact in the middle. Formation of the homo- or heterodimer TLR5 gains human specificity when one residue, Pro268, is supports the hypothesis that dimerization of the ECDs positions mutated to its human counterpart, alanine and vice versa. the cytoplasmic TIR domains close enough to dimerize and Sequence analysis predicts that Pro268 lies on the ascending/ initiate a downstream signaling cascade (Jin et al., 2007; Kang convex surface of LRR9, an atypical LRR motif (Bell et al., et al., 2009; Liu et al., 2008; Park et al., 2009). In most cases,

two ECDs cradle a single ligand molecule, except for TLR4 where two MD-2 molecules each bind a ligand. Most known TLR structures do not use the concave beta-sheet surface for ligand binding, a characteristic mode of binding for other LRR proteins. TLR4 is an exception because it binds its adaptor protein MD-2 in the concave surface. In all TLR complexes, the TLR-ECDs are related by an approx- imate 2-fold symmetry axis. In the TLR 1, 2, 3, and 6 complexes, the nonglycosylated surface of two TLR molecules sandwich a ligand molecule, whereas in the TLR4 complex the coreceptor molecule MD-2 binds the ligand directly. TLR3 binds its ligand mainly by hydrogen bonding and electrostatic interactions whereas hydrophobic interactions dominate the ligand binding in other known TLR structures. The buried surface area in the TLR1/TLR2 interaction is similar to that in the TLR3 homodimer. In the TLR3/dsRNA complex the protein-protein interactions occur only at the LRR-CT, whereas direct interactions between TLR1/TLR2 or TLR2/TLR6 occur near the binding pockets. In the TLR3/dsRNA and TLR4/MD-2/LPS complexes the two C-terminal residues are 25 A˚ apart, whereas in the TLR1/2 and TLR2/6 complexes they are 40 A˚ apart. This larger distance may be due to the fact that the native LRR-CT motifs were replaced with hagﬁsh VLRs, however. In the TLR4/MD-2/ LPS complex, despite the close proximity of the C-termini there is no direct interaction between the two molecules in this region. In the known TLR-ligand complexes, ligands bind TLRs by different mechanisms, but in each case, the ligand bridges two TLR-ECDs on the same glycan-free surface and forms dimers with similar overall architecture. Whether this paradigm will apply to the TLR7-9 family remains to be determined.

The TIR Domain The TLRs signal pathogen attack by dimerization of their cytoplasmic TIR domains in response to ligand-induced dimerization of the ectodomains (Figure 7A). TIR dimerization of TLRs is recognized by TIR domains on the adaptor proteins MyD88, MAL, TRIF, and TRAM, which then trigger downstream signaling pathways, leading to the expression of inflammatory cytokines, various antiviral and antipathogen proteins and to initiation of the adaptive immune response (Figure 7B). The TIR domain is one of many evolutionarily conserved building blocks of the immune system (Figure S1)(Palsson-McDermott and O’Neill, Figure 7. TLR3 Signaling Complex and Signaling Pathways (A) Structural model of the full-length TLR3/dsRNA signaling complex. The 2007). model is based on the mTLR3/dsRNA structure (3CIY) and a TLR3 TIR domain The crystal structures of isolated TIR domains have been re- homology model based on the TLR10 TIR structure (2J67). The trans- ported for TLRs 1, 2 (Xu et al., 2000) and 10 (Nyman et al., membrane portions have been modeled as a helices. (B) TLR signaling pathways. TLR3 signals exclusively through TRIF, whereas 2008)(Table 2), IL-1RAPL (Khan et al., 2004) and for TIR domains TLR4 can use both the TRIF and the MyD88 pathways. All other TLRs use the from Arabidopsis thaliana (Chan et al., 2010) and Paracoccus MyD88 pathway. denitrificans (Chan et al., 2009). In addition, an NMR structure has been reported for the MyD88 TIR domain (Ohnishi et al., 2009). In all TIR domains, alternating b strands and a helices is one of the essential points of interaction in the dimeric inter- are arranged as a central five-stranded parallel b sheet surface, which also contains residues from the DD-loop, and the rounded by five a helices. a C-helix. In addition to being involved in TIR dimer formation, The TIR domains from both TLR1 and TLR2 exist as mono- the BB-loop is sufficiently exposed to interact with adaptor mers in the crystal. Despite sharing 50% sequence identity there proteins during signal transduction. In TLR4, the P681H poly- are important conformational differences between the two struc- morphism, which lies in the BB-loop, abolishes signaling in tures. By contrast, the 2.2 A˚ structure for the TIR domain of response to LPS (Poltorak et al., 1998), indicating that the BB human TLR10 revealed a 2-fold symmetric dimer that has been loop plays an essential role in TLR signaling. The structures of taken to represent the signaling dimer for the TIRs (Figure 8) the plant (Chan et al., 2010) and bacterial TIR domains (Chan (Nyman et al., 2008). The BB-loop that joins strands bB and aB et al., 2009) are similar to their mammalian counterparts.

and IRAK2 (Lin et al., 2010) showed that these molecules form a left handed helical structure containing in order 6 MyD88, 4 IRAK4, and 4 IRAK2 death domains. The MyD88-IRAK4 oligomeric assembly—also called the myddosome—was also conﬁrmed by cryo EM and small angle X-ray scattering experiments (Motshwene et al., 2009). There are three types of interactions observed between the death domains in the myddosome. SNPs in the human MyD88 death domain, S34Y and R98C interfere with the myddosome assembly and may drastically contribute to susceptibility to infection (George et al., 2011). If each TLR-TIR dimer binds two MyD88 TIR domains then the large myddosome superhelix could possibly bridge several acti- vated receptor dimers into a network (Gay et al., 2011). These scaffolds might include more than one type of MyD88-dependent TLR, enabling synergistic responses to microbial stimuli. There are other cases where death domains assemble in superhelical oligomeric signaling complexes like the death-inducing signaling complex (Scott et al., 2009) or the PIDDosome (Park et al., 2007).

Conclusions TLRs are germ-line encoded pattern recognition receptors that initiate defensive responses against a wide variety of pathogens. TLRs are type I membrane receptors with mainly planar, horseshoe-shaped LRR ECDs. TLR-ECD-ligand complex structures are known for TLR1/TLR2 and TLR6/TLR2 with lipopeptides, TLR3 with dsRNA, and TLR4/MD-2 with LPS. Although the Figure 8. Structure of the TIR Domain Dimer from TLR10 TLR-ligand interactions are very different, they all produce an (A) Ribbon diagram of the dimeric TIR10, with the two interacting BB-loops highlighted in gold and red (Nyman et al., 2008) (2J67). m-shaped dimeric complex with C termini in the middle, and (B) Molecular surface of dimeric TIR10. N-termini on the outside. Pairing of the ECD C-termini can lead to dimerization of the cytoplasmic TIR domains, which is recognized homotypically by adaptor molecule TIR domains. MyD88, However, in the latter a dimer is formed that is very different from the most commonly used adaptor, also has death domains that the TLR10 dimer. The BB loops are not involved in dimerization interact with IRAK kinase death domains to form a superhelical contacts but are highly exposed on the surface. An important myddosome, which presumably initiates the signaling cascade. question left in TLR structural biology is the orientation of TIR Despite substantial progress in our understanding of the struc- domains that activate the signaling cascade. Available TIR tural basis of TLR recognition and signaling, several questions domain structures lack the region immediately following their need to be addressed. There is still no structural data for TLRs transmembrane (TM) segment, making it harder to predict their 5, 7, 8, and 9. Amino acid sequences of the TLR 7-9 family exact orientation. In the TLR10 TIR dimer, the N-termini of both suggest that they may differ from other TLRs in both structure molecules have to simultaneously point toward the membrane, and mode of ligand recognition. How TLR-TIR domains interact to accommodate the linker between the TM segment and the with each other or with TIRs from adaptor molecules is still first N-terminal residue from the TIR. unclear. Structures of adaptor molecules would provide insight Purified TIR domains from the MAL and MyD88 adaptor into how they translate TIR recognition into IRAK activation. proteins have been shown to form stable heterodimers in solu- Better understanding of the structural basis for PAMP recognition (Dunne et al., 2003). Dunne et al. (2003) also produced tion and signaling by TLRs could lead to the development of models of the TIR domains from human TLR4, MAL, and adjuvants that specifically bind to TLR-ECDs and activate MyD88 and used these to model the interactions between pairs TLRs or of anti-inflammatory drugs that block TLR mediated of these TIR domains. In these models, the BB loop does not signaling. seem to be involved in either TIR dimerization or in heterotypic interactions with TIRs from the adaptor proteins, which is SUPPLEMENTAL INFORMATION contrary to expectation. Clearly, more structures of interacting TIR domains will be required to better understand how TIR Supplemental Information includes one figure and can be found with this domains function. In addition to its TIR domain, MyD88 contains article online at doi:10.1016/j.str.2011.02.004. a death domain. After binding to TLR TIR domains, MyD88 death domains can interact with the death domains of members of the ACKNOWLEDGMENTS IRAK family of Ser/Thr kinases, triggering their activation and This work was supported by the Intramural Research Program of the National initiating downstream signaling cascades. A structure of a ternary Institutes of Health (NIDDK and NCI) and by a National Institutes of Health/ complex formed by the death domains of human MyD88, IRAK4, Food and Drug Administration intramural biodefense award from NIAID.

REFERENCES Gay, N.J., and Gangloff, M. (2007). Structure and function of Toll receptors and their ligands. Annu. Rev. Biochem. 76 , 141–165. Akashi-Takamura, S., and Miyake, K. (2008). TLR accessory molecules. Curr. Gay, N.J., Gangloff, M., and O’Neill, L.A. (2011). What the Myddosome struc- Opin. Immunol. 20, 420–425. ture tells us about the initiation of innate immunity. Trends Immunol. 32, Andersen-Nissen, E., Smith, K.D., Bonneau, R., Strong, R.K., and Aderem, A. 104–109. (2007). A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin. J. Exp. Med. 204, 393–403. George, J., Motshwene, P.G., Wang, H., Kubarenko, A.V., Rautanen, A., Mills, T.C., Hill, A.V., Gay, N.J., and Weber, A.N. (2011). Two human MYD88 variants, Asagiri, M., Hirai, T., Kunigami, T., Kamano, S., Gober, H.J., Okamoto, K., S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly. J. Biol. 286 Nishikawa, K., Latz, E., Golenbock, D.T., Aoki, K., et al. (2008). Cathepsin K- Chem. , 1341–1353. dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319, 624–627. Govindaraj, R.G., Manavalan, B., Lee, G., and Choi, S. (2010). Molecular modeling-based evaluation of hTLR10 and identification of potential ligands 5 Bell, J.K., Mullen, G.E., Leifer, C.A., Mazzoni, A., Davies, D.R., and Segal, D.M. in Toll-like receptor signaling. PLoS ONE , e12713. (2003). Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol. 24, 528–533. Han, S.H., Kim, J.H., Martin, M., Michalek, S.M., and Nahm, M.H. (2003). Pneumococcal lipoteichoic acid (LTA) is not as potent as staphylococcal Bell, J.K., Botos, I., Hall, P.R., Askins, J., Shiloach, J., Segal, D.M., and Davies, LTA in stimulating Toll-like receptor 2. Infect. Immun. 71, 5541–5548. D.R. (2005). The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc. Natl. Acad. Sci. USA 102, 10976–10980. Han, B.W., Herrin, B.R., Cooper, M.D., and Wilson, I.A. (2008). Antigen recognition by variable lymphocyte receptors. Science 321, 1834–1837. Bella, J., Hindle, K.L., McEwan, P.A., and Lovell, S.C. (2008). The leucine-rich repeat structure. Cell. Mol. Life Sci. 65, 2307–2333. Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R., Yi, E.C., Goodlett, D.R., Eng, J.K., Akira, S., Underhill, D.M., and Aderem, A. (2001). The innate immune Beutler, B., and Rietschel, E.T. (2003). Innate immune sensing and its roots: the response to bacterial flagellin is mediated by Toll-like receptor 5. Nature story of endotoxin. Nat. Rev. Immunol. 3, 169–176. 410, 1099–1103.

Brinkmann, M.M., Spooner, E., Hoebe, K., Beutler, B., Ploegh, H.L., and Kim, He, X.L., Bazan, J.F., McDermott, G., Park, J.B., Wang, K., Tessier-Lavigne, Y.M. (2007). The interaction between the ER membrane protein UNC93B and M., He, Z., and Garcia, K.C. (2003). Structure of the Nogo receptor ectodo- TLR3, 7, and 9 is crucial for TLR signaling. J. Cell Biol. 177, 265–275. main: a recognition module implicated in myelin inhibition. Neuron 38, 177–185. Burch-Smith, T.M., and Dinesh-Kumar, S.P. (2007). The functions of plant TIR domains. Sci. STKE 2007, e46. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000). A Toll-like Chan, S.L., Low, L.Y., Hsu, S., Li, S., Liu, T., Santelli, E., Le, N.G., Reed, J.C., receptor recognizes bacterial DNA. Nature 408, 740–745. Woods, V.L., Jr., and Pascual, J. (2009). Molecular mimicry in innate immunity: crystal structure of a bacterial TIR domain. J. Biol. Chem. 284, 21386–21392. Huizinga, E.G., Tsuji, S., Romijn, R.A., Schiphorst, M.E., de Groot, P.G., Sixma, J.J., and Gros, P. (2002). Structures of glycoprotein Ibalpha and its complex Chan, S.L., Mukasa, T., Santelli, E., Low, L.Y., and Pascual, J. (2010). The with von Willebrand factor A1 domain. Science 297, 1176–1179. crystal structure of a TIR domain from Arabidopsis thaliana reveals a conserved 19 helical region unique to plants. Protein Sci. , 155–161. Janeway, C.A., Jr., and Medzhitov, R. (2002). Innate immune recognition. Annu. Rev. Immunol. 20, 197–216. Choe, J., Kelker, M.S., and Wilson, I.A. (2005). Crystal structure of human toll- 309 like receptor 3 (TLR3) ectodomain. Science , 581–585. Jin, M.S., Kim, S.E., Heo, J.Y., Lee, M.E., Kim, H.M., Paik, S.G., Lee, H., and Lee, J.O. (2007). Crystal structure of the TLR1-TLR2 heterodimer induced by DeLano, W.L. (2002). The PyMOL Molecular Graphics System (San Carlos, CA: binding of a tri-acylated lipopeptide. Cell 130, 1071–1082. DeLano Scientiﬁc). Jung, D., and Alt, F.W. (2004). Unraveling V(D)J recombination; insights into Deng, L., Velikovsky, C.A., Xu, G., Iyer, L.M., Tasumi, S., Kerzic, M.C., Flajnik, gene regulation. Cell 116, 299–311. M.F., Aravind, L., Pancer, Z., and Mariuzza, R.A. (2010). A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey. Proc. Natl. Kajava, A.V. (1998). Structural diversity of leucine-rich repeat proteins. J. Mol. Acad. Sci. USA 107, 13408–13413. Biol. 277, 519–527. Derewenda, U., Li, J., Derewenda, Z., Dauter, Z., Mueller, G.A., Rule, G.S., and Kang, J.Y., Nan, X., Jin, M.S., Youn, S.J., Ryu, Y.H., Mah, S., Han, S.H., Lee, Benjamin, D.C. (2002). The crystal structure of a major dust mite allergen Der H., Paik, S.G., and Lee, J.O. (2009). Recognition of lipopeptide patterns by p 2, and its biological implications. J. Mol. Biol. 318, 189–197. Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 31, 873–884. Divanovic, S., Trompette, A., Atabani, S.F., Madan, R., Golenbock, D.T., Visin- tin, A., Finberg, R.W., Tarakhovsky, A., Vogel, S.N., Belkaid, Y., et al. (2005). Khan, J.A., Brint, E.K., O’Neill, L.A., and Tong, L. (2004). Crystal structure of the 279 Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor Toll/interleukin-1 receptor domain of human IL-1RAPL. J. Biol. Chem. , homolog RP105. Nat. Immunol. 6, 571–578. 31664–31670.

Divanovic, S., Trompette, A., Petiniot, L.K., Allen, J.L., Flick, L.M., Belkaid, Y., Kim, H.M., Park, B.S., Kim, J.I., Kim, S.E., Lee, J., Oh, S.C., Enkhbayar, P., Madan, R., Haky, J.J., and Karp, C.L. (2007). Regulation of TLR4 signaling and Matsushima, N., Lee, H., Yoo, O.J., and Lee, J.O. (2007). Crystal structure of the host interface with pathogens and danger: the role of RP105. J. Leukoc. the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130 Biol. 82, 265–271. , 906–917.

Donnelly, M.A., and Steiner, T.S. (2002). Two nonadjacent regions in enteroag- Kim, Y.M., Brinkmann, M.M., Paquet, M.E., and Ploegh, H.L. (2008). UNC93B1 gregative Escherichia coli ﬂagellin are required for activation of toll-like delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, receptor 5. J. Biol. Chem. 277, 40456–40461. 234–238.

Dunne, A., Ejdeback, M., Ludidi, P.L., O’Neill, L.A., and Gay, N.J. (2003). Struc- Kobe, B., and Deisenhofer, J. (1995). A structural basis of the interactions tural complementarity of Toll/interleukin-1 receptor domains in Toll-like recep- between leucine-rich repeats and protein ligands. Nature 374, 183–186. tors and the adaptors Mal and MyD88. J. Biol. Chem. 278, 41443–41451. Kumar, H., Kawai, T., and Akira, S. (2009). Pathogen recognition in the innate Erridge, C. (2010). Endogenous ligands of TLR2 and TLR4: agonists or assis- immune response. Biochem. J. 420, 1–16. tants? J. Leukoc. Biol. 87, 989–999. Latz, E., Verma, A., Visintin, A., Gong, M., Sirois, C.M., Klein, D.C., Monks, Ewald, S.E., Lee, B.L., Lau, L., Wickliffe, K.E., Shi, G.P., Chapman, H.A., and B.G., McKnight, C.J., Lamphier, M.S., Duprex, W.P., et al. (2007). Ligand- Barton, G.M. (2008). The ectodomain of Toll-like receptor 9 is cleaved to induced conformational changes allosterically activate Toll-like receptor 9. generate a functional receptor. Nature 456, 658–662. Nat. Immunol. 8, 772–779.

Leonard, J.N., Ghirlando, R., Askins, J., Bell, J.K., Margulies, D.H., Davies, Park, B.S., Song, D.H., Kim, H.M., Choi, B.S., Lee, H., and Lee, J.O. (2009). The D.R., and Segal, D.M. (2008). The TLR3 signaling complex forms by coopera- structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. tive receptor dimerization. Proc. Natl. Acad. Sci. USA 105, 258–263. Nature 458, 1191–1195.

Lin, S.C., Lo, Y.C., and Wu, H. (2010). Helical assembly in the MyD88-IRAK4- Peter, M.E., Kubarenko, A.V., Weber, A.N., and Dalpke, A.H. (2009). Identifica- IRAK2 complex in TLR/IL-1R signalling. Nature 465, 885–890. tion of an N-terminal recognition site in TLR9 that contributes to CpG-DNA- mediated receptor activation. J. Immunol. 182, 7690–7697. Liu, L., Botos, I., Wang, Y., Leonard, J.N., Shiloach, J., Segal, D.M., and Davies, D.R. (2008). Structural basis of toll-like receptor 3 signaling with Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van, H.C., Du, X., Birdwell, D., double-stranded RNA. Science 320, 379–381. Alejos, E., Silva, M., Galanos, C., et al. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, Matsumoto, F., Saitoh, S., Fukui, R., Kobayashi, T., Tanimura, N., Konno, K., 2085–2088. Kusumoto, Y., Akashi-Takamura, S., and Miyake, K. (2008). Cathepsins are required for Toll-like receptor 9 responses. Biochem. Biophys. Res. Commun. Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, 367, 693–699. H., Ulmer, A.J., Zahringer, U., Seydel, U., Di, P.F., et al. (1994). Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8, Matsushima, N., Tanaka, T., Enkhbayar, P., Mikami, T., Taga, M., Yamada, K., 217–225. and Kuroki, Y. (2007). Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics 8, 124. Roach, J.C., Glusman, G., Rowen, L., Kaur, A., Purcell, M.K., Smith, K.D., Hood, L.E., and Aderem, A. (2005). The evolution of vertebrate Toll-like recep- Matzinger, P. (1994). Tolerance, danger, and the extended family. Annu. Rev. tors. Proc. Natl. Acad. Sci. USA 102, 9577–9582. Immunol. 12, 991–1045. Schatz, D.G., and Spanopoulou, E. (2005). Biochemistry of V(D)J recombina- Means, T.K., Golenbock, D.T., and Fenton, M.J. (2000). The biology of Toll-like tion. Curr. Top. Microbiol. Immunol. 290, 49–85. receptors. Cytokine Growth Factor Rev. 11, 219–232. Scott, F.L., Stec, B., Pop, C., Dobaczewska, M.K., Lee, J.J., Monosov, E., Medzhitov, R. (2007). Recognition of microorganisms and activation of the 449 Robinson, H., Salvesen, G.S., Schwarzenbacher, R., and Riedl, S.J. (2009). immune response. Nature , 819–826. The Fas-FADD death domain complex structure unravels signalling by receptor clustering. Nature 457, 1019–1022. Miao, E.A., Andersen-Nissen, E., Warren, S.E., and Aderem, A. (2007). TLR5 and Ipaf: dual sensors of bacterial flagellin in the innate immune system. Smith, K.D., Andersen-Nissen, E., Hayashi, F., Strobe, K., Bergman, M.A., Semin. Immunopathol. 29, 275–288. Barrett, S.L., Cookson, B.T., and Aderem, A. (2003). Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and Miura, Y., Shimazu, R., Miyake, K., Akashi, S., Ogata, H., Yamashita, Y., 4 Narisawa, Y., and Kimoto, M. (1998). RP105 is associated with MD-1 and bacterial motility. Nat. Immunol. , 1247–1253. transmits an activation signal in human B cells. Blood 92, 2815–2822. Takeda, K., Kaisho, T., and Akira, S. (2003). Toll-like receptors. Annu. Rev. 21 Miyake, K., Yamashita, Y., Hitoshi, Y., Takatsu, K., and Kimoto, M. (1994). Immunol. , 335–376. Murine B cell proliferation and protection from apoptosis with an antibody Tao, X., Xu, Y., Zheng, Y., Beg, A.A., and Tong, L. (2002). An extensively asso- against a 105-kD molecule: unresponsiveness of X-linked immunodeficient ciated dimer in the structure of the C713S mutant of the TIR domain of human B cells. J. Exp. Med. 180, 1217–1224. TLR2. Biochem. Biophys. Res. Commun. 299, 216–221. Miyake, K., Shimazu, R., Kondo, J., Niki, T., Akashi, S., Ogata, H., Yamashita, Y., Miura, Y., and Kimoto, M. (1998). Mouse MD-1, a molecule that is physically Teghanemt, A., Zhang, D., Levis, E.N., Weiss, J.P., and Gioannini, T.L. (2005). Molecular basis of reduced potency of underacylated endotoxins. J. Immunol. associated with RP105 and positively regulates its expression. J. Immunol. 175 161, 1348–1353. , 4669–4676.

Motshwene, P.G., Moncrieffe, M.C., Grossmann, J.G., Kao, C., Ayaluru, M., Uematsu, S., and Akira, S. (2009). Immune responses of TLR5(+) lamina prop- 44 Sandercock, A.M., Robinson, C.V., Latz, E., and Gay, N.J. (2009). An oligo- ria dendritic cells in enterobacterial infection. J. Gastroenterol. , 803–811. meric signaling platform formed by the Toll-like receptor signal transducers Uff, S., Clemetson, J.M., Harrison, T., Clemetson, K.J., and Emsley, J. (2002). MyD88 and IRAK-4. J. Biol. Chem. 284, 25404–25411. Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain Nyman, T., Stenmark, P., Flodin, S., Johansson, I., Hammarstrom, M., and reveals an unmasking mechanism for receptor activation. J. Biol. Chem. 277 Nordlund, P. (2008). The crystal structure of the human toll-like receptor 10 , 35657–35663. cytoplasmic domain reveals a putative signaling dimer. J. Biol. Chem. 283, 11861–11865. Velikovsky, C.A., Deng, L., Tasumi, S., Iyer, L.M., Kerzic, M.C., Aravind, L., Pancer, Z., and Mariuzza, R.A. (2009). Structure of a lamprey variable lympho- 16 O’Neill, L.A., and Bowie, A.G. (2007). The family of ﬁve: TIR-domain-containing cyte receptor in complex with a protein antigen. Nat. Struct. Mol. Biol. , adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364. 725–730.

Ohnishi, H., Tochio, H., Kato, Z., Orii, K.E., Li, A., Kimura, T., Hiroaki, H., Vijay-Kumar, M., Aitken, J.D., Carvalho, F.A., Cullender, T.C., Mwangi, S., Kondo, N., and Shirakawa, M. (2009). Structural basis for the multiple interac- Srinivasan, S., Sitaraman, S.V., Knight, R., Ley, R.E., and Gewirtz, A.T. tions of the MyD88 TIR domain in TLR4 signaling. Proc. Natl. Acad. Sci. USA (2010). Metabolic syndrome and altered gut microbiota in mice lacking Toll- 106, 10260–10265. like receptor 5. Science 328, 228–231.

Ohto, U., Fukase, K., Miyake, K., and Satow, Y. (2007). Crystal structures of Visintin, A., Iliev, D.B., Monks, B.G., Halmen, K.A., and Golenbock, D.T. (2006). human MD-2 and its complex with antiendotoxic lipid IVa. Science 316, MD-2. Immunobiology 211, 437–447. 1632–1634. Wang, Y., Liu, L., Davies, D.R., and Segal, D.M. (2010). Dimerization of Toll-like Omueti, K.O., Mazur, D.J., Thompson, K.S., Lyle, E.A., and Tapping, R.I. (2007). receptor 3 (TLR3) is required for ligand binding. J. Biol. Chem. 285, 36836– The polymorphism P315L of human toll-like receptor 1 impairs innate immune 36841. sensing of microbial cell wall components. J. Immunol. 178, 6387–6394. Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J.L., and Tong, L. Palsson-McDermott, E.M., and O’Neill, L.A. (2007). Building an immune (2000). Structural basis for signal transduction by the Toll/interleukin-1 system from nine domains. Biochem. Soc. Trans. 35, 1437–1444. receptor domains. Nature 408, 111–115.

Park, H.H., Logette, E., Raunser, S., Cuenin, S., Walz, T., Tschopp, J., and Wu, Yang, D., Tewary, P.,, de la Rosa, G., Wei, F., and Oppenheim, J.J. (2010). The H. (2007). Death domain assembly mechanism revealed by crystal structure of alarmin functions of high-mobility group proteins. Biochim. Biophys. Acta the oligomeric PIDDosome core complex. Cell 128, 533–546. 1799, 157–163.

Park, B., Brinkmann, M.M., Spooner, E., Lee, C.C., Kim, Y.M., and Ploegh, H.L. Yoon, S.I., Hong, M., Han, G.W., and Wilson, I.A. (2010). Crystal structure of (2008). Proteolytic cleavage in an endolysosomal compartment is required for soluble MD-1 and its interaction with lipid IVa. Proc. Natl. Acad. Sci. USA activation of Toll-like receptor 9. Nat. Immunol. 9, 1407–1414. 107, 10990–10995.