Tlr and Pamp

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Dong Hyun Song

Jie-Oh LeeSensing of microbial molecularpatterns by Toll-like receptors

Authors’ addresses

Dong Hyun Song1, Jie-Oh Lee1,2

1Department of Chemistry, KAIST, Daejon, Korea.2Graduate 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: [email protected]

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.

Summary: Toll-like receptors (TLRs) sense structural patterns in micro-bial molecules and initiate immune defense mechanisms. The structuresof many extracellular and intracellular domains of TLRs have beenstudied in the last 10 years. These structures reveal the extraordinarydiversity 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 commonactivation mechanism for TLRs: ligand binding to extracellular domainsinduces dimerization of the intracellular domains and so activates intra-cellular signaling pathways. Recently, the structure of the death domaincomplex of one of the signaling adapters, myeloid differentiation factor88 (MyD88), has been determined. This structure shows how aggrega-tion of signaling adapters recruits downstream kinases. However, weare still far from a complete understanding of TLR activation. We needto study the structures of TLR7 – 10 in complex with their ligands. Wealso need to determine the structures of TLR-adapter aggregates tounderstand activation mechanisms and the specificity of the signalingpathways. 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 activateimmune 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 recognitionreceptors’ (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

Immunological Reviews 2012

Vol. 250: 216–229

Printed in Singapore. All rights reserved

© 2012 John Wiley & Sons A/SImmunological Reviews0105-2896

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2012 John Wiley & Sons A/S216 Immunological Reviews 250/2012


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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 humanTLRs 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

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- andheteromultimers 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

Binding

TLR4-MD-2

Dimerization

Adaptor-Kinase

recruitment

Receptor

clustering (?)

TLR signaling complex

MyD88 IRAK4 IRAK2

MALTLR_TIR

LPS

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

LRRCT

LRRNT

Convex

Concave

Lxx

Lxxx

L

N

N

C

Hydrophobiccore

Intracellular

TIR domain

Extracellular

LRR domain

A s c e n d

i n g l a t e r a l

D e s c e n d i n g l a t e r a l

Transmembranehelix

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

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Song & Lee Structure of Toll-like receptors


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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 allreceptors 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 areexposed 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

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 310

helices, polyproline II helices, or irregular loops. Theirsequence 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 310 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, 310 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 domainsIntracellular 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,

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28). They share substantial structural homology with Ca

rms differences of less than 2 Å. 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 aB (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 bD with helixaD 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

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 aB helix becomes much shorter

because some of the N-terminal residues of the helix are

unwound to be part of the BB loop. The aD 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 TLR2and 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 Ca rms deviation is greater than 2 Å.

Furthermore, the aB and aD helices are changed to irregular

Fig. 3. TIR domain structures. The PdTIR, AtTIR, and L6 TIR domains are from the proteins of Paracoccus denitrificans, Arabidopsis thaliana, and Linumusitatissimum, 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. TheBB and DD loops important in TLR signaling are shown in red. The AtTIR and L6 TIR domains have additional helices in the aD region.

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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 bridgeswith 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 aD 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-raycrystallography (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 asecond 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

TLR2 TLR1 TLR2 TLR6 TLR3

TLR4 TLR5

Triacyl l ipopeptide Diacyl lipopeptide dsRNA

FlagellinLPS

Fig. 4. Structures of Toll-like receptor (TLR)-ligand complexes. The transmembrane helices and intracellular domains are not included in thecrystal structures. Synthetic lipopeptides that contain the di- or triacylated cysteine and several peptide residues were used in the TLR2-TLR1 andTLR2-TLR6 crystallographic studies. The 11 C-terminal modules of TLR5 are deleted in the crystal structure.

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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

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

TLR2 TLR6

Triacyl lipopeptide

Diacyl lipopeptide

TLR3

dsRNA

TLR4

TLR5

Flagellin

D1

D2

D3D3

D2

D3D3

MD-2 MD-2

TLR2 TLR1LRRNT

LRRCT

N-terminal

Central

C - t e r m i n a l Dimerization

interfacePrimary

interface

NN

CC

Triacyl lipoprotein Diacyl lipoprotein

Cys Cys

Protein Protein

NHO

NH3+

S

O

O

O

O

NHO

NH

OS

O

O

O

O

O specific

chain

LPS

Core

Lipid A

O

O

O

O

P-O

O-

O

O O

HO HO

P

O

O-

O-

OO

OHN

O

HO

O NH

O

OO

O

O

A

B

LPS

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 shownby 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 thatdivide 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.

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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-

mannoheptose (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 hydrophobicligands 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

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 hydrophobicinterface. 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). Lipoproteinsare 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

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connected to the N-terminal NH2 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 9 – 12.

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-TLR6complex 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

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 roughlytwo 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 requiredto 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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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 byx-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

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 severalb 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.

Plasma membrane

dsRNATriacyl

lipoprotein

Diacyl

lipoprotein LPSFlagellin

ssRNAUnmethylated

CpG DNA

MAL

T L R 1

T L R 2

MAL

T L R 2

T L R 6

T L R 5

T L R 5

MAL

T L R 4

T L R 4

M D - 2

M D - 2

T L R 3

T L R 3

TIR

TIRTIR

TIR

TIRTIR

TIRTIR

TLR4-MD-2TLR4-MD-2LPS

MyD88

IRAK4 death domain

IRAK2 death domain

MAL

Myddosome

B

MAL

A

TRIF

Endosome

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 andadapters are not known and are shown schematically as boxes.

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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 havesimilar 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 onlya 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-dependentsignaling 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

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 thestructures 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 –

TIRinteraction 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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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 layershave 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-bindingprotein (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

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: CD14transfers 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

BPI CD14

RP105-MD-1

N N

C C

RP105 RP105

A

C

B

Fig. 7. Structures of proteins involved in Toll-like receptor

activation. Structures of BPI (A), CD14 (B), and the RP105-MD-1complex (C). CD14 forms a homodimer. The monomeric subunits of CD14 are colored in green and blue, respectively. The MD-1 moleculesbound to RP-105s are schematically drawn in grey.

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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 1 – 6 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 structuralrearrangements 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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