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Dong Hyun Song Jie-Oh Lee Authors’ addresses Dong Hyun Song 1 , Jie-Oh Lee 1

Dong Hyun Song Jie-Oh Lee

Authors’ addresses Dong Hyun Song 1 , Jie-Oh Lee 1,2 1 Department of Chemistry, KAIST, Daejon, Korea. 2 Graduate School of Nanoscience & Technology (WCU), KAIST, Daejon, Korea.

Correspondence to:

Jie-Oh Lee Department of Chemistry, Graduate School of Nanoscience & Technology (WCU), KAIST Daejon, Korea Tel.: +82 42 350 2839 Fax: +82 42 350 2810 e-mail: jieoh@kaist.ac.kr

Acknowledgement We gratefully acknowledge the support of the World Class University Program (R31 10071) of the Ministry of Education, Science and Technology of Korea. The authors have no conflicts of interest to declare.

This article is part of a series of reviews covering Insights from Structure appearing in Volume 250 of Immunological Reviews .

Immunological Reviews 2012 Vol. 250: 216–229 Printed in Singapore. All rights reserved

© 2012 John Wiley & Sons A/S

Immunological Reviews

0105-2896

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Sensing of microbial molecular patterns by Toll-like receptors

Summary: Toll-like receptors (TLRs) sense structural patterns in micro- bial molecules and initiate immune defense mechanisms. The structures of many extracellular and intracellular domains of TLRs have been studied in the last 10 years. These structures reveal the extraordinary diversity of TLR-ligand interactions. Some TLRs use internal hydropho- bic pockets to bind bacterial ligands and others use solvent-exposed surfaces to bind hydrophilic ligands. The structures suggest a common activation mechanism for TLRs: ligand binding to extracellular domains induces dimerization of the intracellular domains and so activates intra- cellular signaling pathways. Recently, the structure of the death domain complex of one of the signaling adapters, myeloid differentiation factor 88 (MyD88), has been determined. This structure shows how aggrega- tion of signaling adapters recruits downstream kinases. However, we are still far from a complete understanding of TLR activation. We need to study the structures of TLR710 in complex with their ligands. We also need to determine the structures of TLR-adapter aggregates to understand activation mechanisms and the specificity of the signaling pathways. Ultimately, we will have to study the structures of the com- plete TLR signaling complexes containing full-length receptors, ligands, signaling, and bridging adapters, and some of the downstream kinases to understand how TLRs sense microbial infections and activate immune responses against them.

Keywords: Toll-like receptor , innate immunity , pattern recognition , MyD88

Toll-like receptors

Toll-like receptors (TLRs) provide efficient and immediate immune responses to bacterial, fungal, and viral infections by recognizing diverse molecules released from them. Because they recognize common patterns in microbial mole- cules, they are often referred to as ‘pattern recognition receptors’ (1, 2). Humans have 10 TLRs, and each of them binds a distinct family of microbial molecules (3, 4). TLR2 forms a heterodimer with either TLR1 or TLR6 and binds bacterial lipoproteins. TLR4 in complex with MD-2 recog- nizes the lipopolysaccharide (LPS) that is a major compo- nent of the outer membrane of Gram-negative bacteria. TLR5 can be stimulated by a depolymerized form of flagel- lin. TLR3 and TLR7 9 respond to various forms of bacterial or viral nucleic acid. The engagement of TLRs with ligand

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

molecules induces dimerization of the receptors, which leads to recruitment of intracellular signaling adapters and kinases. The activation of TLR signaling pathways eventually results in the synthesis of pro-inflammatory cytokines and therefore prepares the entire immune system for infection. TLRs are important targets for drug design because excess activity of TLRs is involved in various inflammatory diseases (5). Antagonists of TLR4 are being developed as targets of anti-sepsis drugs (6 9); agonists of TLR7 and TLR8 are being used as anti-viral and anti-cancer drugs, and many more are in clinical trials (5). A partial agonist of TLR4 has recently been approved as a general vaccine adjuvant, and agonists targeted to several TLRs are being developed for this purpose (10 12).

Overview of the TLR activation mechanism

Like other single transmembrane receptors, TLRs are acti- vated by ligand-induced multimerization ( Fig. 1 ). The struc- tures of the key intermediates in these activation processes have been mainly studied by x-ray crystallography (13 21). The extracellular domain of each TLR has the characteristic horseshoe-like structure of the leucine-rich repeat (LRR) family of proteins (13, 14, 22, 23). The concave surface consists of a large central b sheet, and the convex surface is decorated by a combination of helices and irregular loops ( Fig. 2 ). Binding of agonistic ligands to TLRs induces dimer- ization of their extracellular domains. We now have struc- tures for the extracellular domains of six of the 10 human TLRs in the ligand-induced dimeric state. Although the ligand recognition mechanisms of the TLRs vary substan- tially, all the TLR dimers have a similar overall arrangement:

the C-termini of the two extracellular domains converge in the middle, separated by distances ranging from 20 to 40 angstrom. This structural observation has led to the proposal that ligand binding to the extracellular domains enhances

Song & Lee Structure of Toll-like receptors

LRRNT LRRCT
LRRNT
LRRCT
& Lee Structure of Toll-like receptors LRRNT LRRCT helix Convex C Hydrophobic N core N L

helix

Convex C Hydrophobic N core N L L L x x x x x Concave
Convex
C
Hydrophobic
N
core
N
L
L
L
x
x
x
x
x
Concave
Ascending lateral
Descending lateral

Extracellular

LRR domain

Transmembrane

Intracellular

TIR domain

Fig. 2. Overall structure of a Toll-like receptor (TLR). The extracellular and intracellular domains of TLR4 are drawn as a blue surface. A C a trace of one of the LRR modules and the side chains of the conserved leucines and asparagine is shown. The LRRNT and LRRCT modules are colored pink and green, respectively.

dimerization of the intracellular domains because the latter are connected to the C-termini of the extracellular domains by transmembrane helices (15). The intracellular domains of TLRs, as well as of several signaling adapters, share a domain named the Toll/interleukin-1 receptor (TIR) domain. Its structure is composed of a parallel b sheet sur- rounded by a helices (24 29). Ligand-induced aggregation of the receptors and adapters is mediated by homotypic and heterotypic interaction between these TIR domains. Although the structures of quite a few monomeric TIRs have already been reported, the structures of their homo- and heteromultimers remain unsolved. All these structural studies have been conducted with truncated extracellular or intracellular domains because the full-length receptors including the transmembrane helices are difficult to produce and crystallize. Therefore, the struc- tural information of the full-length TLRs under membrane embedded condition is not available. There is evidence that

LPS TLR signaling complex LPS TLR4-MD-2 Binding Dimerization Adaptor-Kinase Receptor TLR_TIR MAL recruitment
LPS
TLR signaling complex
LPS
TLR4-MD-2
Binding
Dimerization
Adaptor-Kinase
Receptor
TLR_TIR
MAL
recruitment
clustering (?)
MyD88
IRAK4
IRAK2

Fig. 1. Overall mechanism of TLR4 activation. The plasma membrane is shown schematically by pink horizontal bars. The TLR4 extracellular domains are shown as black arcs and the MD-2s as green balls.

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

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Song & Lee Structure of Toll-like receptors

activated and dimerized TLRs form higher order clusters in the cell membrane (25, 30). However, structures of these proposed clusters have never been characterized in detail because of difficulties reconstituting the complete receptor signaling complexes that includes the full-length receptors, ligands, signaling, and bridging adapters and some of the downstream kinases. This is a common problem for all receptors with single transmembrane helices, and future research in receptor biology should address this fundamental problem.

Structures of the monomeric TLRs without bound ligands

The extracellular domains

The available x-ray structures of the extracellular domains of TLRs in the monomeric state reveal the conformations of inactive TLRs prior to binding ligand (14, 17, 31). In addi- tion to this, several structures of TLR2 and TLR4 bound to non-agonistic or antagonistic ligands have been determined (16, 17). The extracellular domains of TLRs belong to the LRR family. The amino acid sequences of LRR family pro- teins are composed of multiple copies of repeat modules named LRR modules (22, 23) (Fig. 2). Each module is 20 30 amino acids long and has a characteristic LxxLxLxxN sequence motif. The central LxL part of the module forms the core of a b strand. The two leucines point towards the interior of the protein, making up the hydrophobic core, whereas the variable 9 residues within the motif are exposed to solvent, and some are involved in interactions with ligands. The conserved asparagines in the motifs form

a continuous hydrogen bonding network with backbone

carbonyl oxygens in neighboring LRR modules throughout the protein. The overall structures of the LRR proteins resemble a horseshoe. The b strands provided by the each LRR module assemble into a large b sheet making up the entire concave surface of the horseshoe. Variable amino acids outside the conserved LRR motif of each module gen- erate the convex part of the structure. The LRR modules are

protected by two special modules named LRRNT and LRRCT

in the N- and C-termini of the proteins. These modules do

not conform to the sequence conservation pattern of the LRR modules and often contain an anti-parallel b hairpin stabilized by disulfide bridges. The majority of the LRR family proteins are involved in protein protein interactions, and their protein-binding sites are located in their concave surfaces. TLRs are atypical members of the LRR family: with the exception of TLR5, their ligands are not proteins and

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their ligand-binding sites are not located in their concave surfaces (4). The LRR family is divided into seven subfamilies, each of which has a different module length and sequence conserva- tion pattern in the convex part of the structure (22, 23). Some of them have parallel a helices, and others have 3 10 helices, polyproline II helices, or irregular loops. Their sequence conservation patterns suggest that the TLRs belong to the ‘typical’ subfamily (17, 32, 33). However, they devi- ate substantially from the canonical pattern of the subfamily. The LRR modules of the typical subfamily contain 24 amino acids and have 3 10 helices in the convex area. On the other hand, the lengths of TLR modules vary between 20 and 28 amino acids, and the convex parts of their structures are composed of mixtures of a helices, 3 10 helices, and irregular loops. Because of this, the convex surface of a TLR is rough rather than smooth and contains multiple grooves and shal- low pockets that can serve for ligand interaction. Further- more, the conserved asparagines are missing in some of the LRR modules in lipid-interacting TLRs, TLR1, TLR2, TLR4, and TLR6; this allows unusual distortions of the horseshoe- like structures, giving rise to sharp transitions in the torsion and twist angles of the central b sheet (4, 15, 17). Such structural distortion permits the formation of hydrophobic pockets in TLR1 and TLR2 and also has a role in the interac- tion of TLR4 with MD-2.

Structures of the intracellular domains

Intracellular TIR domains are found not only in the TLRs but also in the adapters involved in TLR signaling pathways (34, 35). Five signaling and bridging adapters, MyD88, MyD88-adapter-like protein (MAL), TIR-domain-containing adapter-inducing interferon- b (TRIF), translocating chain- associating membrane protein (TRAM), and sterile-a and Armadillo motif-containing protein (SARM), have been found to be important in human TLR responses. MAL, TRIF, and TRAM are also called TIR-domain-containing adapter protein (TIRAP), TIR-containing adapter molecule-1 (TI- CAM-1), and TICAM-2, respectively. All TIR domains have a similar overall structure ( Fig. 3 ), with a central five-stranded parallel b -sheet surrounded by five a helices (24, 26 29). Sequence conservation in the TIR family is relatively low, in the 20 30% range, and domain lengths vary between 135 and 160 residues. This is because of large deletions and insertions in the loop regions. The structures of monomeric TIR domains have been determined in the cases of TLR1, TLR2, and TLR10 (26,

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

Song & Lee Structure of Toll-like receptors

Song & Lee Structure of Toll-like receptors Fig. 3. TIR domain structures. The PdTIR, AtTIR, and

Fig. 3. TIR domain structures. The PdTIR, AtTIR, and L6 TIR domains are from the proteins of Paracoccus denitrificans , Arabidopsis thaliana , and Linum usitatissimum , respectively. The figure was drawn using coordinate files with PDB codes, 1FYV (TLR1), 1FYW (TLR2), 2J67 (TLR10), 2Z5V (MyD88), 2Y92 (MAL), 1T3G (IL-1R), 3H16 (PdTIR), 3JRN (AtTIR), and 3OZI (L6). The a helices are green and the central b sheet is blue. The BB and DD loops important in TLR signaling are shown in red. The AtTIR and L6 TIR domains have additional helices in the a D region.

28). They share substantial structural homology with C a

˚

rms differences of less than 2 A . Mutagenesis studies have identified several regions that are important for signaling activity. The most prominent region identified is the BB loop that connects strand bB and helix a B (26, 28, 36 39). The BB loop extends away from the rest of the TIR domain, forming a protrusion from the surface. A P712H substitu- tion in TLR4 abolishes the host response to LPS. The corre- sponding residue in TLR2 is located in the BB loop and does not have any clear structural role, suggesting that it is functionally but not structurally important. The TLR10 TIR domain has been crystallized as a homodimer with an extensive dimer interface, although it exists as a monomer in solution (26). The dimerization interface is symmetrically shaped, involves the BB loop, and shows substantial sequence conservation. Because of this, the dimeric arrange- ment of TLR10 TIR has been used as the template for TIR homodimerization in several modeling studies (38). Some mutations in the DD loop that connects strand b D with helix a D also have significant effects on TLR activation, suggesting that the DD loop is part of the TIR TIR interaction interface along with the BB loop (26, 28, 36 39). The structures of the TIR domains of the signaling adapt- ers MyD88 and MAL have also been reported (27, 29). Human MyD88 consists of 296 amino acids and is com- posed of two signaling domains. The C-terminal TIR domain mediates interaction with the receptor, and the N- terminal death domain is responsible for recruiting the

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

IRAK4 and IRAK1/2 kinases that initiate downstream signal-

ing. The structure of the MyD88 TIR domain has been determined by nuclear magnetic resonance (NMR) spectros- copy (27). Its overall structure is similar to those of the TLR TIR domains. The largest structural difference is in the BB and DD loop regions; the a B helix becomes much shorter because some of the N-terminal residues of the helix are unwound to be part of the BB loop. The a D helix is com- pletely changed to an irregularly shaped loop. Using a NMR-based method, they have performed binding studies between the MyD88 and MAL TIR domains. They found that these two TIR domains interact weakly, with a Kd value of approximately 7 micromoles. Alanine substitution of R196 of MyD88 reduced MAL binding twofold, and mutations of the residues R288 and R217 also substantially reduced MAL binding. These observations suggest that the continuous interface containing the BB loop and these arginine residues mediates the TIR TIR interaction with MAL. MAL is required for MyD88-dependent signaling by TLR2 and TLR4 (34, 35). Other TLRs do not need MAL for MyD88-dependent signaling. Human MAL has 221 amino acid residues that make up a phosphatidyl inositol-binding motif and a TIR domain. Although the TIR domain has a similar overall fold composed of a central b sheet sur- rounded by a helices, its structure is rather different from other TLR TIR domains (29). Its sequence identity is in the

˚

low twenties and the C a rms deviation is greater than 2 A . Furthermore, the a B and a D helices are changed to irregular

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Song & Lee Structure of Toll-like receptors

loops. Cysteines play important roles in the structure and function of MAL, forming two disulfide bridges, C89 C134 and C142 C174. Disulfide bridges are rare in intracellular proteins, although not unprecedented. The two other cyste- ines, C91 and C157, are not involved in forming disulfide bridges with protein residues. However, they appear to be chemically reactive and form simultaneous disulfide bridges with a single DTT molecule in the buffer. Mutagenesis experiments have shown that these cysteines are crucial for MyD88 binding. Interestingly, the C157 position is highly conserved in the TLR family and the corresponding residue in TLR4 is the target site of TAK-242 (40). This is an exper- imental drug that blocks TLR4 signaling by inhibiting its interaction with MAL and TRAM (9). TIR domains are found not only in the mammalian pro- teins but also in several bacterial and plant proteins. The bacterial TIR-containing proteins are thought to interfere with the host immune system by hijacking MyD88 through direct TIR TIR interaction (41 44). Plants contain a large number of related proteins which are involved in resistance to infection (45, 46). These proteins contain single TIR domains that are thought to be involved in multimerization. The structures of the bacterial TIR domain of Paracoccus deni- trificans and the plant TIR domains of Linum usitatissimum and Arabidopsis thaliana have been determined (36, 47, 48). These non-animal TIR domains have the similar overall fold expected from the sequence conservation, although the two

plant TIRs have additional helices in the a D helix region ( Fig. 3 ).

Dimerization and activation of TLRs

Agonistic ligands induce homo- or heterodimerization of TLR extracellular domains. The structures of these TLR- ligand complexes have been extensively studied by x-ray crystallography (15, 16, 18, 20, 21) ( Fig. 4 ). Their interac- tions with ligands are extraordinarily diverse (Fig. 5A ). TLR2 forms heterodimers with TLR1 and TLR6 after binding lipo- peptides (15, 16). The lipid chains of the ligands are inserted into hydrophobic chambers within TLR2 and TLR1. For binding to the lipid chains of LPS, TLR4 use a hydro- phobic chamber not present in the TLR itself but in the accessory protein MD-2 (17, 19). MD-2 interacts with the N-terminal and central domains of TLR4, and LPS induces dimerization of the TLR4-MD-2 heterodimers, forming het- erotetrameric complexes of two TLRs and two MD-2s via a second TLR4 interaction site in MD-2 (20). Hydrophilic interactions play major roles in ligand recognition by TLR3 and TLR5. TLR3 has two ligand-binding sites located near the N-terminus and C-terminus, respectively, of its extracel- lular domain, and flagellin binds to a concave surface formed by the N-terminal nine LRR modules of TLR5 (18, 21). In each of these cases, the ligands play direct roles in TLR dimerization by interacting simultaneously with two TLRs. All the resulting TLR structures have strikingly similar

Triacyl lipopeptide

Diacyl lipopeptide

dsRNA

TLR2 TLR1 TLR2 TLR6 TLR3 LPS Flagellin
TLR2
TLR1
TLR2
TLR6
TLR3
LPS
Flagellin

TLR4

TLR5

Fig. 4. Structures of Toll-like receptor (TLR)-ligand complexes. The transmembrane helices and intracellular domains are not included in the crystal structures. Synthetic lipopeptides that contain the di- or triacylated cysteine and several peptide residues were used in the TLR2-TLR1 and TLR2-TLR6 crystallographic studies. The 11 C-terminal modules of TLR5 are deleted in the crystal structure.

220

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

A Triacyl lipopeptide

Central N-terminal LRRNT TLR2 TLR1 LRRCT C-terminal
Central
N-terminal
LRRNT
TLR2
TLR1
LRRCT
C-terminal

N

Diacyl lipopeptide N TLR2 TLR6 C C
Diacyl lipopeptide
N
TLR2
TLR6
C
C
dsRNA
dsRNA

TLR3

N Diacyl lipopeptide N TLR2 TLR6 C C dsRNA TLR3 B - O Core O specific
N Diacyl lipopeptide N TLR2 TLR6 C C dsRNA TLR3 B - O Core O specific
N Diacyl lipopeptide N TLR2 TLR6 C C dsRNA TLR3 B - O Core O specific

B

- O

Core

O specific

chain

O O O P O O O HO O - O O P O -
O
O
O
P
O
O
O
HO
O
-
O
O
P
O -
O
-
HN
O
NH
O
O
O
O
O
O
O
HO
HO
O
O

Lipid A

LPS

Song & Lee Structure of Toll-like receptors

LPS MD-2 MD-2 TLR4 D3 D3 D2 D2 Flagellin D1
LPS
MD-2
MD-2
TLR4
D3
D3
D2 D2
Flagellin
D1

TLR5

Dimerization Primary interface interface D3 D3
Dimerization
Primary
interface
interface
D3
D3
Protein Protein O NH O NH Cys Cys NH + NH 3 S O S
Protein
Protein
O
NH
O
NH
Cys
Cys
NH
+
NH 3
S
O
S
O
O
O
O
O
O
O
O
Triacyl lipoprotein
Diacyl lipoprotein

Fig. 5. Ligand induced homo- and heterodimerization of the extracellular domains of Toll-like receptors (TLRs). (A) Diversity of the TLR- ligand interaction. The extracellular domains of TLRs are shown as arcs. The C-terminal fragment of TLR5 deleted in the crystal structure is shown by dashed lines. The D3 domain of flagellin is not visible in the electron density map, presumably because it is very flexible. The boundaries of the LRRNT and LRRCT modules are drawn with solid lines. Some TLRs, namely TLR1, TLR2, TLR4, and TLR6, have structural transitions that divide them into three subdomains, N-terminal, central, and C-terminal. The boundaries of these subdomains are shown by dashed lines. (B) Chemical structures of LPS and lipoproteins.

shapes resembling the letter ‘m’. In these dimeric structures, the two C-termini of the extracellular domains converge in the center while the N-termini stretch outwards (15, 16, 18, 20, 21). This finding suggests the hypothesis that dimerization of the extracellular domains leads to juxtaposi- tion of the intracellular domains, and the signaling adapters are believed to be recruited into such dimerized forms of the TLR intracellular domains (15). The structural changes

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

induced by ligands are minimal, and only minor adaptations of flexible loops or amino acid side chains are involved in ligand binding.

Dimerization of TLR4-MD-2-LPS complexes

The principal ligand of TLR4 is lipopolysaccharide (LPS), which is the main component of the outer membrane of

221

Song & Lee Structure of Toll-like receptors

Gram-negative bacteria (49 51). LPS is a macromolecular glycolipid composed of a hydrophobic lipid A region attached to a long and branched carbohydrate chain ( Fig. 5B ). The lipid A has two glucosamine sugars to which typically four to seven lipid chains are attached (52 54). Lipid A is negatively charged because two phosphate groups are attached to the glucosamine backbone. Significant struc- tural diversity has been reported in the structure of lipid A. For example, the number of lipid chains can vary, and the phosphate groups can be deleted, or modified by several other chemical groups (52, 54). The carbohydrate chain of LPS can be divided into two areas, the core and the O-spe- cific chain (50). The relatively conserved core oligosaccha- ride chain contains multiple copies of the unusual sugars, 3- deoxy-D- manno -oct-2-ulosonic acid (Kdo) and L-glycero-D- manno heptose (Hep). The core region is frequently modified by negatively charged chemical groups such as phosphates. The O-specific chain is composed of many copies of repeat- ing units containing galactoses, galactosamines, and other sugars. The structure and composition of the repeating units differ between bacterial species and even between different growth conditions in the same species. Complete removal of the core and O-specific chain has only a minor effect on the immunological activity of LPS. Therefore, the lipid A region is believed responsible for most of its inflammatory activity. Crystallographic investigations have confirmed this and have shown that hydrophobic interaction of lipid A with a pocket in MD-2 makes the main contribution to LPS binding

(20).

LPS is recognized by TLR4-MD-2 heterodimers expressed on the surface of immune cells (55 57). MD-2 is approxi- mately 18 kDa glycoprotein that functions as the LPS-bind- ing subunit of the TLR4-MD-2 heterodimer (57 60). It has two anti-parallel b sheets sandwiched together, like immu- noglobulin fold proteins. But the disulfide bond connecting the two sheets in the latter is absent from MD-2 (17, 19). Because of this, the two b sheets in the sandwiched struc- ture of MD-2 can be separated to generate a large internal pocket that is ideally shaped for binding flat hydrophobic ligands like LPS. MD-2 has two TLR4 binding sites, named the primary interface and dimerization interface, respectively (17, 20) (Fig. 5A ). The primary interaction interface is formed between one edge of MD-2 and part of the concave surface provided by the N-terminal and central domains of TLR4. This well-conserved interaction interface is mostly composed of ionic and hydrogen bonds and does not require binding of LPS. The dimerization interface of MD-2 is located opposite the primary interface and interacts with a

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convex surface provided by a small hydrophobic patch in the C-terminal domain of TLR4. One partially exposed lipid chain of LPS is located in the core of the dimerization inter- face. This lipid chain and surrounding hydrophobic amino acid residues of MD-2 interact with the hydrophobic patch of TLR4. Several hydrophilic interactions between protein residues of MD-2 and TLR4 support this core hydrophobic interface. The two phosphate groups of lipid A form multi- ple charge and hydrogen bond interactions with both MD-2 and the two TLR4s and therefore contribute to the stability of the dimerization interface. The structure-activity relationships of LPS have been stud- ied for decades using natural and chemically modified LPS (50 52, 61). The results show that the total number of lipid chains and the presence of the two phosphate groups in lipid A are the most important factors affecting its inflam- matory activity. The crystal structure provides an explanation of this observation (20). The six lipid chains are optimal because partial exposure of one of the lipid chains is critical for forming the dimerization interface. When the LPS has less than six lipid chains, part of the MD-2 binding pocket becomes empty, and this will destabilize the overall interac- tion (62). When the LPS has more than six lipid chains, the additional lipid chain may interfere with the interaction between TLR4 and MD-2 because it is likely to prevent the approaching TLR4 from achieving the optimal interaction distance. The phosphate groups are essential because they provide the charge and hydrogen bonding network. Dele- tion of either of these phosphates reduces inflammatory activity more than a 100-fold (54, 61, 63). A lipid A deriv- ative with one of the phosphate groups deleted retains more or less, all of its immune stimulatory activity, but loses most of its inflammatory toxicity. Because of this, it has recently been approved as a general vaccine adjuvant (10).

Formation of TLR2-TLR1 and TLR2-TLR6-lipoprotein complexes

TLR2 forms heterodimers with TLR1 or TLR6 and interacts with bacterial lipoproteins (15, 16, 64, 65). Lipoproteins are a family of proteins that are anchored to the bacterial membrane by lipid chains covalently attached to conserved N-terminal cysteines (66 70) ( Fig. 5B ). The protein parts of the lipoproteins have little sequential and functional homol- ogy. The lipoproteins found in the Gram-negative bacteria have three lipid chains; two of them are connected to the glycerol group, which is in turn attached to the sulfur atom of the N-terminal cysteine. These lipid chains are referred to as ‘ester-bound’ lipid chains. The third lipid chain is

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

connected to the N-terminal NH 2 via an amide bond. There- fore, it is referred to as an ‘amide-bound’ lipid chain. The lipoproteins and lipopeptides of Gram-positive bacteria and mycoplasmas were thought to have only two lipid chains because they lack the enzyme responsible for attaching the amide-bound lipid chain (71 73). However, this view has recently been challenged because several lipoproteins puri- fied from Staphylococcus aureus were shown to have three lipid chains (74, 75). In addition to lipoproteins, many bacterial and fungal molecules have been proposed to function as agonistic ligands for TLR2, and many, but not all, of these candidate ligands also contain diacylglycerol groups (3, 4). TLR2-TLR1 heterodimers are the principal receptors for triacylated lipoproteins (65). The three lipid chains of the ligand bridge the TLRs by interacting simultaneously with TLR2 and TLR1 via the three lipid chains; two lipid chains are inserted into the large hydrophobic pocket in TLR2, and the third, the amide-bound chain, is inserted into the narrow hydrophobic channel in TLR1 (15) ( Fig. 5A ). The entry of the TLR2 pocket lies near the boundary between the central and C-terminal domains and extends into an internal hydrophobic pocket formed by LRR modules 912. The glycerol and the peptide backbone atoms of the two N-terminal residues of the lipoprotein form extensive hydrogen bonds with amino acids in both TLR2 and TLR1. Direct TLR1 TLR2 interaction also contributes to the stabil- ity of the heterodimer. The core of the interface is formed by several hydrophobic residues. It is surrounded by hydro- philic residues forming ionic and hydrogen bonds. The Pro315 SNP of TLR1 frequently found in Caucasian popula- tions appears to block TLR1 signaling by perturbing this heterodimerization interface (15, 76). Diacylated lipoproteins from Gram-positive bacteria or mycoplasmas mainly activate TLR2-TLR6 heterodimers (64). The two ester-bound lipid chains are inserted into the same TLR2 pocket as TLR2-TLR1 triacyl lipopeptide complexes, and the hydrophilic glycerols and peptide backbones of the lipoproteins form hydrogen bonds with amino acid residues of both TLR2 and TLR6 (16) ( Fig. 5A ). The TLR2-TLR6 complex cannot bind to the triacylated lipoproteins because the hydrophobic channel responsible for interaction with the amide-bound lipid chain in TLR1 is blocked by two bulky phenylalanines in TLR6. Mutation of these residues into the ones found in TLR1 renders TLR6 capable of responding to both diacylated and triacylated lipoproteins. In the TLR2-TLR1-triacylate lipopeptide complex, the amide-bound lipid chain plays an important role in TLR heterodimer formation. Without the amide-bound lipid

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

Song & Lee Structure of Toll-like receptors

chain in the diacylated lipopeptide, TLR2 and TLR6 can still form stable heterodimers because the size of the hydro- phobic core of the protein protein interface is increased, and this can compensate for the missing lipid channel interaction.

Homodimerization of the TLR3-dsRNA complex

The principal ligand of TLR3 is the double-stranded RNA that can be generated during virus replication (3). The receptor is localized mainly in endocytic organelles and its activation requires TRIF instead of MyD88 (Fig. 6A ). The crystal structure of the complex between the TLR3 extracel- lular domain and a 46 bp double-stranded RNA has been determined by x-ray crystallography (18). TLR3 has two RNA binding sites located in its N-terminal and C-terminal

regions, respectively (Fig. 5A ). The distance between the two

˚

sites is approximately 70 A , which corresponds to roughly two helical turns of RNA. The backbone phosphates and sugars of dsRNA make major contributions to TLR3 binding at both sites. Ligand binding and TLR3 dimerization require an acidic environment below pH 6.0 (77). The TLR3 struc- ture can explain this strong pH dependence because several histidines make crucial bonds with the phosphate backbones of the RNA, and their protonation states appear essential for stability of the interaction. The nucleic acid bases make min- imal contact with the TLR, which accounts for the sequence-independent binding of RNA. Although approxi- mately 40 bp of RNA appears to be the minimum required to induce TLR3 dimerization, a robust immune response requires RNA of more than approximately 100 bp. Further- more, binding of RNA by TLR3 shows strong positive coo- perativity (77). These observations suggest that clustering of more than one TLR3 dimer may be necessary for an ade- quate immune response. A recent study supports this model (30). They produced antibodies that can block activity of TLR3. Interestingly, two of these antibodies did not directly interfere with neither of the two ligand-binding sites. Based on crystallographic and molecular modeling study, they pro- posed that these antibodies interrupt TLR3 activation by inhibiting lateral clustering of the TLR3 dimers.

Homodimerization of the TLR5-flagellin complex

Flagellae are large protein complexes that confer motility on bacteria. They are made up of multiple copies of approxi- mately 20 different protein subunits (78, 79). Electron microscopy shows that they consist of three substructures, a basal body generating torque, a hook connecting the basal

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Song & Lee Structure of Toll-like receptors

A

Triacyl Diacyl Flagellin Unmethylated dsRNA ssRNA lipoprotein lipoprotein LPS CpG DNA Plasma membrane MAL MAL
Triacyl
Diacyl
Flagellin
Unmethylated
dsRNA
ssRNA
lipoprotein
lipoprotein
LPS
CpG DNA
Plasma membrane
MAL
MAL
MAL
Endosome
TRIF
B
TLR4-MD-2
TLR4-MD-2
LPS
TIR
TIR
MAL
MAL
TIR
TIR
TIR
TIR
MyD88
TIR
TIR
IRAK4 death domain
IRAK2 death domain
TLR1
TLR2
TLR2
TLR6
TLR5
TLR5
MD-2
TLR4
TLR4
MD-2
TLR3
TLR3

Myddosome

Fig. 6. Toll-like receptor (TLR) signaling complexes. (A) A variety of signaling and bridging adapters are recruited to active TLRs. (B) Proposed structure of the TLR4-MD-2-LPS-Myddosome complex. The structures of the multimerized TIR complexes between receptors and adapters are not known and are shown schematically as boxes.

body to a helical fiber, and the long flagellin fiber. The latter is assembled from a large number of fliC flagellins. A high- resolution structure of a fliC fragment has been determined by x-ray crystallography (80). To obtain monomeric proteins suitable for crystallization, the D0 domain formed by the N- terminal and C-terminal fragments was removed by proteoly- sis and the resulting approximately 45 kD fragment contain- ing the remaining middle domains, D1, D2, and D3, was used for structure analysis. The highly conserved D1 domain plays the main role in polymerization of the fliC subunits into a helical fiber. It is composed of three long a helices and a b hairpin. Two of the helices are formed by the N-terminal part

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of the protein, and the third helix by the C-terminal region. The helices and the b hairpin are arranged in an anti-parallel bundle-like structure. The D2 domain is composed of several b hairpin structures and connects the D1 and D3 domains. The D3 domain is formed by the middle part of fliC, and its sequence is highly diverse in different bacterial species. It is exposed to the surface of the flagellin fiber and mediates the reversible lateral interactions between flagellin fibers. Because neither vertebrates nor plants contain flagellae, the presence of these structures serves as an early sign of bacterial infection in both classes of organism (81). TLR5 is the major innate immune receptor for flagellin in vertebrates.

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

Recently, the crystal structure of zebrafish TLR5 has been determined in a complex with a truncated fragment of Salmonella fliC (21). As with TLR2 and TLR4, several hybrid proteins consisting of TLR fragments fused with VLR proteins from jawless fishes were used for crystallization (15 17). The TLR5 of fish and humans have substantial sequence homology, suggesting that the two proteins have similar structures and ligand recognition mechanisms. The structure obtained shows that the flagellin D1 domain plays the dominant role in binding and dimerization of TLR5 ( Fig. 5A ). One face of the D1 domain interacts with an extended surface area encompassing the LRR modules from LRRNT to LRR9 of TLR5. At the same time, the D1 domain forms a bond with the second TLR5 molecule in the dimer and therefore bridges the two TLRs. The overall shape and curvature of the horseshoe-like structure formed differ in only minor ways from those of TLR5 without bound ligand. This observation applies to all TLR structures, thus underlin- ing the high structural rigidity of the LRR fold. The spatial arrangement of the two TLR5 molecules in the dimer also resembles closely that of the other TLR dimers. The two C- terminal regions join in the center and the two N-termini splay outwards. Because the TLR5 fragment that they used for crystallization does not contain native TLR sequences beyond LRR14, it is not clear from the structure alone if the C-terminal part of the receptor contributes to dimerization or to ligand interaction. However, mutagenesis experiments suggest that the deleted C-terminal region probably has only a minor role (21).

Dimerization of intracellular domains and recruitment of signaling adapters

Binding of agonistic ligands initiates intracellular signaling by recruiting specific adapters to the intracellular TIR domains of TLRs ( Fig. 6A ). In humans, TLR activation involves five proteins, MyD88, MAL, TRIF, TRAM, and SARM (34, 35). MyD88 is required for signaling by all TLRs except TLR3. The latter uses TRIF instead of MyD88. The bridging adapter, MAL, is necessary for MyD88-dependent signaling by TLR2 and TLR4. The MyD88-MAL-dependent signaling by TLR4 is activated mainly on the plasma mem- brane, whereas TRIF-TRAM-dependent signaling by TLR4 is activated on endocytic organelles (82). TRAM is the bridg- ing adapter for TRIF. It is necessary for signaling by TLR4 but not by TLR3. SARM negatively regulates TLR signaling by interacting with TRIF and blocking its function (83). All these adapters contain TIR domains that are essential for sig- nal activation. Mutagenesis experiments have shown that

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

Song & Lee Structure of Toll-like receptors

recruitment of adapters to receptor TIRs is mediated by TIR TIR interactions. In addition to TIR domains, MAL contains an N-terminal phosphatidyl inositol-binding motif and TRAM has a myristoylation site (84, 85). These additional motifs are necessary for membrane localization of these bridging adapters. An important aim of research has been to determine the structures of TIR homo- and heterocomplexes. However, the TLR TIR domains produced as truncated forms by genetic manipulation interact only weakly or not at all with the TIR domains of signaling adapters (28). This makes the direct structural study of the TIR complexes very difficult. Hence, identification of interaction interfaces has been attempted by site-directed mutagenesis. Ronni et al. (86) introduced approximately 70 alanine substitution and short deletion mutations into the human TLR4 TIR domain and measured the effect on the IL-7 response in transfected HEK293 cells. To differentiate the signal of the introduced TLR4 from the endogenous TLR4, they used a constitutively active chimeric receptor with the extracellular domain of CD40 and the intracellular domain of TLR4. Using this chimeric receptor, they found that mutations clustered in two regions, the BB and DD loops, resulted in substantial attenuation of the TLR4 signaling. However, it is not clear whether these regions are involved in the homotypic aggregation of the TLR TIRs or the heterotypic interactions with adapter TIR domains. Recently, Bovijn et al. (87) advanced research on TIR TIR interaction by identifying mutations that specifically affect homotypic interactions of TLR4 TIR domains using a tech- nique called MAPPIT, which detects protein interactions in mammalian cells using chimeric receptors. They discovered several interesting aspects of TIR interaction. First, the MyD88-TLR4 interaction requires as bridging adapter, the MAL TIR domain. Second, the TIR domains of the bridging adapters MAL and TRAM can interact directly with the TLR4 TIR domain without the help of signaling adapters, MyD88 or TRIF. Third, they found that the regions surrounding the BB loops are crucial for TLR4 TIR homomultimerization. Finally, they also found that all mutations that affect the MAL or TRAM interaction block TLR4 TIR multimerization, suggesting that TLR4 TIR multimerization is a prerequisite for the MAL/TRAM interaction. In addition to the TIR domain, MyD88 contains a death domain that is required for activation of downstream effec- tors. The downstream kinases that are directly recruited to the TLR-MyD88 complex are IRAK4 and IRAK1/IRAK2. Lin et al. (25) reported a crystal structure of the death domain

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Song & Lee Structure of Toll-like receptors

complex of MyD88-IRAK4-IRAK2 ( Fig. 6B ). The TIR domain of MyD88 and kinase domains of IRAKs were removed for the crystallization. These workers found that the death domains form a large helical complex that they have named as ‘Myddosome’. In this complex, six MyD88, four IRAK4, and four IRAK2 death domains are arranged as a left-handed helix composed of four distinct layers. The top two layers have six MyD88s, the middle layer, four IRAK4s, and the bottom layer, four IRAK2s. Some deletion or substitution mutations that cause life threatening bacterial infections mapped in the binding interface, demonstrating an indis- pensable role for the complex in TLR signaling. Based on this structure, it was proposed that formation of the Myddo- some brings the kinase domains of the IRAKs into proximity so that they can be phosphorylated and activated. Because the Myddosome complex has six MyD88 death domains, it appears that the complete TLR signaling com- plex contains more than one set of TLR dimers. If we assume that the TIR domain of MyD88 interacts 1:1 with the TLR TIR, then six MyD88 molecules in the Myddosome are capable of binding three TLR dimers ( Fig. 6B ). This kind of higher order clustering of receptor multimers has been proposed for quite a few membrane receptors (30, 88 92). However, characterization of high-resolution structures of the clusters has not been possible, mostly due to difficulties in making full-length receptors and reconstituting complete signaling complexes in a membrane environment.

Structures of functionally and structurally related proteins, LBP, CD14, RP105, and MD-1

LPS and lipoproteins are amphipathic macromolecules, and they form aggregated micelle structures in water solution. Because of this, efficient extraction of these molecules from bacterial membrane requires accessary proteins, lipopolysac- charide-binding protein (LBP), and CD14. LBP is an acutely induced plasma protein that binds avidly to LPS and delivers it to CD14 (93). Addition of purified LBP enhances the sensitivity of macrophages to LPS 100- to 1000-fold (94). LBP belongs to the lipid transfer/lipopolysaccharide-binding protein (LT/LBP) family (95). Other members of the family include bactericidal/permeability-increasing protein (BPI), cholesterol ester transfer protein (CETP), phospholipid transfer protein (PLTP), and a few poorly characterized pro- teins. LBP and BPI share 48% sequence identity and bind to and regulate the biological effects of LPS. BPI is a boomer- ang-shaped protein formed by two domains of similar size connected by a proline-rich linker (96) ( Fig. 7A ). Each domain has a deep hydrophobic pocket that can bind

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phospholipids on the concave surface of the boomerang. As the sequences of LBP and BPI can be aligned over their entire lengths, LBP is expected to share the general structural features of BPI: its elongated shape and pseudosymmetry as well as the three structural elements, the two barrels, central sheet, and apolar phospholipid-binding pockets. However, the two proteins have clear functional difference: CD14 transfers LPS to TLR4-MD-2, but BPI cannot. This difference need to be explained by future structural study of LBP. CD14 is an LRR family protein that is involved in the transfer of LPS from LBP to the TLR4-MD-2 complex. It is expressed on the surface of myelomonocytic cells as a gly- cosylphosphatidylinositol (GPI)-linked glycoprotein or in soluble form in serum (97). In addition to the LPS of Gram-negative bacteria, CD14 can bind other amphipathic microbial products, such as peptidoglycan, lipoteichoic acid, lipoarabinomannan, and lipoproteins (98, 99). The mono- meric subunit of CD14 contains 11 LRR modules and a sin- gle LRRNT module (100) ( Fig. 7B ). CD14 does not contain an LRRCT module, which is required to cover the hydro- phobic core of the protein from exposure to solvents. Instead, CD14 uses the exposed hydrophobic core to form a homodimer. Each CD14 monomer contains an N-terminal hydrophobic pocket that is formed between the LRRNT and the first LRR modules. The convex surface between these

A

the first LRR modules. The convex surface between these A C BPI RP105 RP105 N N

C

BPI

RP105

RP105

N N C C
N
N
C
C

RP105-MD-1

B

between these A C BPI RP105 RP105 N N C C RP105-MD-1 B CD14 Fig. 7.

CD14

Fig. 7. Structures of proteins involved in Toll-like receptor activation. Structures of BPI (A), CD14 (B), and the RP105-MD-1 complex (C). CD14 forms a homodimer. The monomeric subunits of CD14 are colored in green and blue, respectively. The MD-1 molecules bound to RP-105s are schematically drawn in grey.

© 2012 John Wiley & Sons A/S Immunological Reviews 250/2012

Song & Lee Structure of Toll-like receptors

two modules is cracked open, exposing the hydrophobic core residues inside. The size of the pocket is large enough to accommodate the lipid chains of a single LPS molecule. Therefore, the function of CD14 appears to be monomer- ization of the aggregated ligands for efficient binding to

TLR4.

RP105 and MD-1 are structurally and sequentially homol- ogous to TLR4 and MD-2, respectively (101 103). How- ever, unlike TLR4, RP105 does not contain a sizable intracellular domain, suggesting it cannot activate signaling pathways by itself. The exact function and the physiological ligands for the RP105-MD-1 complex have not been clearly identified yet, although it has been proposed that this pro- tein complex regulates TLR2 and TLR4 signaling by directly interacting with them (104 107). Several structures of the RP105-MD-1 complex have recently been determined (108 111) ( Fig. 7C ). The individual structures of RP105 and MD- 1 closely resemble those of TLR4 and MD-2, respectively, and the primary interaction interface between RP105 and MD-1 shows a homology with that of TLR4 and MD-2. However, unlike TLR4-MD-2, which requires LPS for the formation of a heterotetramer, the RP105-MD-1 can form a stable heterotetramer without bound ligands. Furthermore, the overall arrangement of the two RP105 in the complex shows a clear difference from the arrangement of TLR4 in the TLR4-MD-2-LPS complex: the N-terminal LRR modules, instead of the C-terminal modules, of the two RP105 chains

converge in the center of the complex ( Fig. 7C ). This head- to-head arrangement of RP105 contrasts with the tail-to-tail mode, which is highly conserved in the ligand-activated TLR homo- and heterodimers ( Fig. 5A ). The structure of the MD-1 pocket is homologous with that of MD-2, suggesting that MD-1 may be able to interact with flat hydrophobic ligands like LPS.

Conclusion

Over the last 10 years, important progress has been made in structural understanding of the ligand recognition and acti- vation mechanisms of TLRs. The structures of the extracellu- lar domains of TLRs 16 have been determined in complex with their cognate ligands. The structures of the intracellular TIR domains of several TLRs and adapters have also been determined. These structures show us how TLRs recognize their ligands and how ligand binding induces structural rearrangements of the receptor. For the next 10 years, the most important goal should be determining structures of the complete signaling complexes comprising the full-length receptors, ligands, signaling adapters, bridging adapters, and downstream kinases. To achieve this ambitious goal, we must first find ways to produce enough amounts of structur- ally and functionally intact receptors and to reconstitute the complete receptor-ligand adapter-kinase complexes from the purified components.

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