Structure

Review

The Structural Biology of Toll-like Receptors

Istvan Botos,1 David M. Segal,2 and David R. Davies1,*1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases2Experimental Immunology Branch, National Cancer InstituteNational Institutes of Health, Bethesda, MD 20892, USA*Correspondence: david.davies@nih.govDOI 10.1016/j.str.2011.02.004

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

IntroductionIn the initial phase of an infection, the innate immune system

generates a rapid inflammatory response that blocks the growth

and dissemination of the infectious agent. This response is

followed, in vertebrates, by the development of an acquired

immune response in which highly specific B and T cell receptors

recognize the pathogen and induce responses that lead to its

elimination (Janeway and Medzhitov, 2002). The antigen recep-

tors of the acquired immune system are well characterized. They

consist of many structurally similar molecules with different

binding specificities created by somatic rearrangements and

mutations within the binding site regions of the B and T cell

receptor variable domains (Jung and Alt, 2004; Schatz and

Spanopoulou, 2005). By contrast, the receptors of the innate

immune system are germline-encoded and have been selected

by evolution to recognize pathogen-derived compounds that

are essential for pathogen survival or endogenous molecules

that are released by the host in response to infection (Matzinger,

1994; Yang et al., 2010; Erridge, 2010). Innate immune receptors,

also known as pattern recognition receptors (PRRs), have been

identified in the serum, on the cell surface, in endosomes, and

in the cytoplasm (Medzhitov, 2007). The Toll-like receptors

(TLRs) represent a particularly important group of PRRs

(Gay and Gangloff, 2007). TLR paralogs are located on cell

surfaces or within endosomes and have important roles in the

host defense against pathogenic organisms throughout the

animal kingdom. In humans, 10 TLRs respond to a variety of

pathogen-associated molecular patterns (PAMPs), including

lipopolysaccharide (TLR4), lipopeptides (TLR2 associated with

TLR1 or TLR6), bacterial flagellin (TLR5), viral dsRNA (TLR3), viral

or bacterial ssRNA (TLRs 7 and 8), and CpG-rich unmethylated

DNA (TLR9), among others (Kumar et al., 2009).

The TLRs are type I integral membrane receptors, eachwith an

N-terminal ligand recognition domain, a single transmembrane

helix, and a C-terminal cytoplasmic signaling domain (Bell

et al., 2003). The signaling domains of TLRs are known as Toll

IL-1 receptor (TIR) domains because they share homology with

the signaling domains of IL-1R family members (O’Neill and

Bowie, 2007). TIR domains are also found in many adaptor

Stru

proteins that interact homotypically with the TIR domains of

TLRs and IL-1 receptors as the first step in the signaling

cascade. Remarkably, homologs of TIR domains are also found

in some plant proteins that confer resistance to pathogens

(Burch-Smith and Dinesh-Kumar, 2007), suggesting that the

TIR domain represents a very ancient motif that served an

immune function before the divergence of plants and animals.

The transmembrane domains of TLRs each contain a typical

stretch of �20 uncharged, mostly hydrophobic residues. TLRs

that recognize nucleic acid PAMPs interact through their trans-

membrane domains with a multispan transmembrane protein

known as UNC93B, which directs these TLRs to endocytic

compartments (Brinkmann et al., 2007; Kim et al., 2008). The re-

maining TLR paralogs do not interact with UNC93B, and traffic

directly to the cell surface. The N-terminal ectodomains (ECDs)

of TLRs are glycoproteins with 550–800 amino acid residues

(Bell et al., 2003). These ectodomains are either extracellular or

in endosomes where they encounter and recognize molecules

released by invading pathogens. We review our current under-

standing of the structural basis for ligand recognition and signal

transduction by TLRs.

Leucine-Rich Repeats—The Building Blocks of TLRsAll TLR ECDs are constructed of tandem copies of a motif known

as the leucine-rich repeat (LRR), which is typically 22–29 resi-

dues in length and contains hydrophobic residues spaced at

distinctive intervals (Figure 1A). This motif is found in many

proteins in animals, plants and microorganisms, including

many proteins involved in immune recognition (Palsson-McDer-

mott and O’Neill, 2007). In three dimensions, all LRRs adopt

a loop structure, beginning with an extended stretch that

contains three residues in the b strand configuration that were

recently reviewed (Bella et al., 2008) (Figure 1B). When assem-

bled into a protein, multiple consecutive LRRs form a solenoid

structure, in which the consensus hydrophobic residues point

to the interior to form a stable core and the b strands align to

form a hydrogen-bonded parallel b sheet. Because the b strands

are more closely packed than the non-b portions of the LRR

loops, the solenoid is forced into a curved configuration in which

cture 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved 447

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

Structure

Review

the concave surface is formed by the b sheet (Kajava, 1998) (Fig-

ure 1C). As a result, each LRR protein contains a concave

surface, a convex surface, an ascending lateral surface that

consists of loops connecting the b strand to the convex surface,

448 Structure 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved

and a descending lateral surface on the

opposite side (Bella et al., 2008).

The first LRR protein structure

described was ribonuclease inhibitor (RI)

(Kobe and Deisenhofer, 1995). The

LRRs in this protein are relatively long,

typically 27–29 amino acids in length,

and all LRRs have three to four turns of

a-helix on their convex surfaces opposite

the b sheet. RI contains 16 LRRs that do

not form a complete circle, but form

a ‘‘horseshoe’’ structure (Figure 1C). In

the innate immune system, a family of

cytoplasmic danger sensors known as

‘‘Nod-like receptors’’ (NLRs) contain

nine or fewer contiguous RI-like LRRs at

their C-terminal ends, which are thought

to be involved in recognizing danger

signals. Based on the RI structure, one

would predict that the LRRs of the NLR

proteins form banana-shaped structures

with a b sheet on their concave surfaces

and a helices on their convex surfaces.

The TLR-ECDs typically contain 19–25

LRRs that, like RI, form horseshoe struc-

tures. In contrast to RI, the consensus

LRR of the TLRs is 24 residues in length

(Figure 1A), which does not allow for the

formation of multi-turn helices on their

convex sides. Consequently, interstrand

distances on the convex side are shorter

for TLRs than for RI, which gives rise to

TLR-ECD structures with lower curvature

and larger outer diameters (�90 A) than

for RI (Figure 1C). The 24-residue

consensus LRRs adopt a variety of configurations on their

convex sides, often containing bits of secondary structure

such as b strands, 310 helices, and polyproline II helices. In other

proteins that contain 24-residue LRRs, for example glycoprotein

Table 1. Main Features of the 10 Human TLR Molecules

TLR Residues LRRsaN-Linked

Glycosylation Sitesb Accession Code

1 786 19 4 (7) Q15399

2 784 19 3 (4) O60603

3 904 23 11 (15) O15455

4 839 21 5 (10) O00206

5 858 20 (7) O60602

6 796 19 8 (9) Q9Y2C9

7 1049 25 (14) Q9NYK1

8 1041 25 (18) Q9NR97

9 1032 25 (18) Q9NR96

10 811 19 (8) Q9BXR5

LRR, leucine-rich repeat; TLR, Toll-like receptors.a The number of LRRs in the extracellular domain do not include the

LRR-NT or LRR-CT motifs.bNumber of N-glycosylation sites observed in the crystal structure or

predicted by the NetNGlyc server 1.0 in parentheses (http://www.cbs.

dtu.dk/services/NetNGlyc/).

Figure 2. The Structure of a TLR-ECD (hTLR3)Top and side views of the TLR3-ECD, with the N-linked glycosyl moieties(Bell et al., 2005) (2A0Z). The LRRs are capped by the LRR-NT and LRR-CTmotifs and themolecule is flat with one glycan-free side (ascending side) that isinvolved in receptor dimerization.

Structure

Review

Iba (Uff et al., 2002), Nogo receptor (He et al., 2003), and the vari-

able lymphocyte receptors (VLRs) of jawless vertebrates among

others, a slight twist relates each LRR to its neighbor, which

generates a nonplanar overall structure. The twist in these

proteins is most likely due to the inherent twist found in b sheets.

By contrast, the known TLR-ECD structures are more planar,

suggesting that structures on the convex and lateral sides of

TLR LRRs counteract the twist tendency on the concave side.

Planarity of TLR-ECDs may be important for ligand binding and

activation (see below).

A characteristic feature of TLR-ECDs is the frequent occurrence

of LRRs that are substantially larger than the consensus 24 resi-

dues, especially in TLRs 7, 8, and 9. These extra residues often

produce loops thatprotrude fromtheTLR-ECDhorseshoe,usually

on the ascending or convex side of the LRR (Figure 1B). The

TLR-ECDs also contain structures that cap the N and C-terminal

ends known as the LRR-NT and LRR-CT motifs, respectively

(Figure 2). The LRR-NTs are disulfide-linked b-hairpins, whereas

the LRR-CTs are globular structures that contain two a helices

and are stabilized by two disulfide bonds. Similar capping motifs

have been observed in several other proteins that contain

24-residue LRRs (He et al., 2003; Huizinga et al., 2002).

In most LRR proteins, ligand binding occurs on the concave

surface (Figure 1D). For example, the VLRs of jawless vertebrates

Stru

generate great diversity by randomly joining LRRs from a large

pool of genomically encoded LRR cassettes into the mature

VLR protein. In these molecules, the highest diversity in amino

acid sequence occurs on the concave b surface. In the two

ligand-VLR structures available, binding occurs on this surface

(Deng et al., 2010; Han et al., 2008; Velikovsky et al., 2009)

(Figure 1E). In addition, a large loop that interacts with the ligand

protrudes from the LRR-CT in both structures, which is also seen

in the glycoprotein Iba-von Willebrand factor complex (Huizinga

et al., 2002) (Figure 1F). By contrast, in the known TLR-ligand

structures, ligand binding occurs most often on the ascending

lateral surface of the TLR-ECD (Jin et al., 2007; Kang et al.,

2009; Liu et al., 2008; Park et al., 2009) (Figure 2). This surface

is the only portion of the molecule that completely lacks N-linked

glycan and is free to interact with a ligand.

The Structure of TLRsBased on sequence homologies, vertebrate TLRs can be

grouped into six subfamilies, TLR1/2/6/10, TLR3, TLR4, TLR5,

TLR7/8/9, and TLR11/12/13/21/22/23 (Matsushima et al.,

2007; Roach et al., 2005). Not all vertebrate species express all

TLR paralogs; humans, for example lack all members of

the TLR11 family. As indicated in Table 1, the ECDs of the

10 human TLRs vary in LRR numbers and extent of N-linked

glycosylation. To date, the structures of the ECDs of TLRs 1, 2,

3, 4, and 6 (human or mouse) have been reported (Table 2). All

ECDs assume the typical horseshoe-shape, but their structures

cannot be superimposed because of variations in curvature.

In the known structures, the glycans are distributed throughout

themolecule, except for the lateral face formed by the ascending

loops of the LRRs (Figure 2). This glycan-free face is involved in

dimerization on ligand binding in the known TLR/ligand struc-

tures (see below).

The TLR1/2/6/10 SubfamilyTLR2 resides on the plasma membrane where it responds to

lipid-containing PAMPs such as lipoteichoic acid and di- and

cture 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved 449

Table 2. TLR-ECD and TIR domain structures

Molecule

Residues

TLR\VLR Resolution A PDB Code Reference

TLR monomers

hTLR2a T1–284\V136–199 1.8 2Z80 Jin et al., 2007

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

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

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

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

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

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

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

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

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

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

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

mTLR4/mMD-2 T27–625 2.8 2Z64 Kim et al., 2007

hMD-2 2.0 2E56 Ohto et al., 2007

hMD-2/lipid IVa 2.2 2E59 Ohto et al., 2007

TLR dimeric complexes

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

T27–506\V133–199

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

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

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

T33–482\V157–232

TIR domains

hTIR1a 2.9 1FYV Xu et al., 2000

hTIR2 3.0 1FYW Xu et al., 2000

hTIR2 P681H mutant 2.8 1FYX Xu et al., 2000

hTIR2 C713S mutant 3.2 1O77 Tao et al., 2002

hTIR10 2.2 2J67 Nyman et al., 2008

TIR (human IL-1RAPL) 2.3 1T3G Khan et al., 2004

TIR (Arabidopsis) 2.0 3JRN Chan et al., 2010

pdTIR (Paracoccus) 2.5 3H16 Chan et al., 2009

TIR (MyD88) NMR 2Z5V Ohnishi et al., 2009

TIR (MyD88) NMR 2JS7 Unpublished

NMR, nuclear magnetic resonance; PDB, Protein Data Bank; PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriamine-

pentaacetic acid (synthetic phospholipid derivative); pnLTA, Streptococcus pneumoniae lipoteichoic acid; TIR, Toll IL-1 receptor; TLR, Toll-like recep-

tors; VLR, variable lymphocyte receptors. Available in the PDB as of 2010.a Human (h) and mouse (m) proteins with the residue numbers shown for the TLR part (T) and the VLR part (V) in case of the chimeric constructs. The

length of the full TLR-ECDs including the signal peptide: TLR1 577 residues, TLR2 585, TLR3 696, TLR4 627, and TLR6 582.

Structure

Review

tri-acylated cysteine-containing lipopeptides (Takeda et al.,

2003). It does this by forming dimeric complexes with either

TLR1 or TLR6 on the plasma membrane. The structures of

TLR2/TLR1 and TLR2/TLR6 with lipopeptide ligands have been

determined (Jin et al., 2007; Kang et al., 2009). To facilitate crys-

tallization and structure determination the LRR-CT and the last

one or two LRRs of TLRs 1, 2, and 6 were replaced by corre-

sponding regions of a hagfish VLR (Table 2) (Kim et al., 2007).

Structural analyses revealed that the ECDs of TLRs 1, 2, and 6

contain three distinctive subdomains: N-terminal, central, and

450 Structure 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserve

C-terminal (Figure 3A). The N-terminal subdomain contains the

LRR-NT capping motif and LRRs 1–4, with typical 24-residue

LRR modules. In this region, two features commonly found in

the interiors of LRR structures are preserved: an asparagine

ladder, formed by a continuous network of hydrogen bonds

between consensus asparagines (Figure 1A) and neighboring

backbone oxygens (Kajava, 1998); and a spine formed by

consecutive consensus phenylalanine residues (Figure 1A) (Jin

et al., 2007). By contrast, the central and C-terminal subdomains

have LRR modules with atypical sequences, and their b sheet

d

Figure 3. Structure of the TLR1/TLR2/Pam3CSK4 Complex(A) The TLR2-ECD showing the position of thethree subdomains: N-terminal, central, andC-terminal.(B) hTLR1 and Pam3CSK4 ligand interactionsmapped on the molecular surface.(C) hTLR2 and Pam3CSK4 interactions mapped onthe surface.(D) Ribbon diagram of TLR1/TLR2 bindingPam3CSK4 (magenta) (Jin et al., 2007) (2Z7X).

Structure

Review

conformations deviate from those seen in standard LRRs. The

central subdomain is also lacking the asparagine ladder and

phenylalanine spine.

The border between the central and C-terminal subdomains

(LRRs 9–12) harbors ligand-binding pockets on the convex

side in both TLR1 and TLR2 ECDs (Figures 3B and 3C). These

pockets are lined with hydrophobic residues and can accommo-

date fairly long lipid chains from lipopeptide ligands. The shapes

of lipid binding pockets vary between species (Jin et al., 2007).

For example, the mouse TLR2 binding pocket is shorter than

the human. As a result, mouse TLR2 binds relatively short lauryl

chains more efficiently than human TLR2, which is also reflected

in the capacity of lauryl3CSK4 to activate mouse, but not human

TLR2. In the TLR1/TLR2 complex (Figure 3D), the triacylated

lipopeptide ligand, Pam3CSK4, bridges the two TLR-ECDs by

inserting the two ester-bound palmitoyl groups into the TLR2

binding pocket, and the single amide-bound palmitoyl chain

into the TLR1 pocket (Jin et al., 2007) (Figures 3B and 4A).

TLRs 1 and 2 also interact with the head group of Pam3CSK4

by forming hydrogen bonds with the glycerol and peptide

portions and by forming hydrophobic interactions with the sulfur

atom. Importantly, TLRs 1 and 2 interact with each other on their

dimerization surfaces via several hydrogen bonds and hydro-

phobic interactions in the vicinity of the binding pockets. The

importance of these interactions to TLR1/2 function is suggested

by the observation that a polymorphic mutation in this region,

P315L, abolishes its ability to respond to lipopeptide ligands

(Omueti et al., 2007). The ligand-protein and protein-protein

interactions bring the C-terminal regions of the ECDs in close

proximity, giving rise to an overall structure of the complex that

has been described as having an ‘‘m’’-shape (Figure 3D). The

close apposition of the C-terminal regions likely facilitates

Structure 19, April 13, 2011

the dimerization of the TIR domains on

the cytoplasmic side of the plasma

membrane.

Whereas the TLR1/2 complex recog-

nizes tri-acylated lipopeptides such as

Pam3CSK4, the TLR2/6 complex recog-

nizes the di-acylated ligand, Pam2CSK4.

The latter ligand lacks the lipid chain

that binds in the hydrophobic pocket of

TLR1, raising the question of how the di-

acylated lipopeptide is able to stabilize

a complex between TLRs 2 and 6. The

crystal structure of the TLR2/TLR6/

Pam2CSK4 complex (Kang et al., 2009)

indicates that TLR6 and TLR1 have very

similar, horseshoe structures and that the TLR2/6 complex

adopts the same overall m-shaped structure as seen in the

TLR1/2 complex (Figure 4B). However, TLRs 1 and 6 contain

important structural differences in their ligand binding and dimer-

ization regions. In TLR6, the side chains of two phenylalanine

residues block the lipid binding channel, leading to a channel

that is less than half the length as that of TLR1 (Figure 4C).

This structural feature provides selectivity for diacylated over

triacylated lipopeptides, as confirmed by the observation that

mutation of the F343 and F365 residues in TLR6 to their TLR1

counterparts allows TLR6 to respond to the triacylated

Pam3CSK4 (Kang et al., 2009). In the TLR2/6 complex, the two

lipid chains of Pam2CSK4 are buried in the TLR2 hydrophobic

pocket as in the TLR1/2 complex whereas the peptide part of

Pam2CSK4 forms several hydrogen bonds with both TLR2 and

TLR6 (Figure 4C). The LRR11 loop in TLR6 is slightly displaced

relative to its position in TLR1 and forms a prominent hydrogen

bond with the carbonyl oxygen of the first peptide bond of the

ligand. In addition, the LRR11-LRR14 regions of TLR2 and

TLR6 form a heterodimeric interface via hydrophobic and hydro-

philic interactions of their surface-exposed residues. The area of

hydrophobic interaction is 80% larger than in the hTLR1/hTLR2

complex, suggesting that this surface interaction together with

the hydrogen bond between LRR11 and the ligand drives the

heterodimerization of TLR6.

Lipoteichoic acid (LTA), a glycolipid from Gram-positive

bacterial membranes contains a diacylated glycerol group con-

nected by an ether bond to a variable carbohydrate moiety.

LTAs vary in their capacity to activate TLR2, for example Staph-

ylococcus aureus LTA is a much more potent activator than

Streptococcus pneumoniae LTA. The crystal structure for

S. pneumoniae LTA bound to the TLR2/VLR hybrid shows

ª2011 Elsevier Ltd All rights reserved 451

Figure 4. Ligand Binding by TLR 1, 2, and 6(A) Cross-section through themolecular surface ofthe TLR1/TLR2/Pam3CSK4 complex (2Z7X).(B) Ribbon diagram of TLR2/TLR6 bindingPam2CSK4 (magenta) (Kang et al., 2009) (3A79).(C) Cross-section through themolecular surface ofthe TLR2/TLR6/Pam2CSK4 complex (3A79).(D) Cross-section through themolecular surface ofthe TLR2/pnLTA complex (Kang et al., 2009)(3A7B).(E) Cross-section through the molecular surface ofthe TLR2/PE-DTPA complex (Kang et al., 2009)(3A7C).

Structure

Review

differences compared to lipopeptide bound to TLR2 (Kang et al.,

2009) (Figure 4D). Curiously, this LTA failed to dimerize TLR2 or

induce hetero-dimers with TLR1 or TLR6, although it was

capable of activating TLR2 in cells, probably by forming

a complex with TLR1 (Han et al., 2003). Another nonpeptidic

ligand PE-DTPA (a synthetic derivative of phosphatidylethanol-

amine) binds mTLR2/VLR similar to LTA (Kang et al., 2009)

(Figure 4E). These TLR2/ligand structures are missing the

hydrogen bonding network present in TLR2-lipopeptide

complexes, due to the shift of the carbohydrate head groups

relative to the peptide head groups of lipopeptide ligands. This

shift is also enhanced by the repulsion between the head group

oxygen atom of the ligand and the hydrophobic sulfur binding

site of TLR2, which is normally filled by the sulfur atom from

the lipopeptide cysteine. The hydrogen bonding network is

possibly required to maintain the correct conformation of the

TLR2 LRR11 loop that makes crucial intermolecular contacts

with TLR1 or TLR6 in the heterodimeric complexes.

There are four different types of protein-ligand interactions for

the TLR 1, 2, and 6 complexes. These interactions can be ranked

in order of their importance: hydrophobic interactions with the

TLR2 pocket, hydrogen bonding of peptide head groups, hydro-

452 Structure 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved

phobic interactions with the TLR1

channel, and hydrophobic interaction of

the conserved cysteine from peptide

headgroups. The heterodimer is further

stabilized by hydrophobic, hydrogen-

bonding, and ionic interactions between

the TLR molecules.

Phylogenic analysis identifies TLR10 as

part of the TLR-1 family (Roach et al.,

2005). Based on the above TLR1/TLR2

and TLR2/TLR6 complex structures,

homology models of hTLR10 complexes

were constructed and refined through

molecular dynamics simulations (Govin-

daraj et al., 2010). The hTLR2/hTLR10

and hTLR1/hTLR10 complex models

were similar to the available TLR1 family

complexes. However, the binding orien-

tation of the hTLR10 homodimer was

different due to the presence of nega-

tively charged surfaces near LRR 11–14

that define a specific binding pocket.

Docking experiments suggest that

Pam3CSK4 might be a ligand for the

hTLR2/hTLR10 complex and PamCysPamSK4 might bind to

hTLR1/hTLR10 and the hTLR10 homodimer.

TLR3TLR3 recognizes dsRNA, which is produced by most viruses at

some stage in their life cycles and is a potent indicator of viral

infection. In contrast to several cytoplasmic receptors for

dsRNA, TLR3 is localized to endosomes where it recognizes

dsRNA. Studies using soluble TLR3-ECD protein (Leonard

et al., 2008) or immobilized TLR3-GFP constructs (Wang et al.,

2010) showed that TLR3-ECD is monomeric in solution but binds

as dimers to 45-bp segments of dsRNA, the minimum length

required for TLR3 binding and activation. In addition, binding is

independent of base sequence and occurs only at pH 6.5 and

below (Leonard et al., 2008). A crystal structure of the TLR3-

ECD dimer complexed with a 46-bp dsRNA oligonucleotide

explains these properties.

The first reported crystal structure of a TLR binding domain

was that of the unliganded form of TLR3-ECD (Bell et al., 2005;

Choe et al., 2005) (Figure 2). The TLR3-ECD horseshoe is largely

uniform and flat, lacking the subdomain structure seen in TLRs 1,

2, 4, and 6. However, irregularities in the LRRs produce short

Figure 5. Structure of the TLR3/dsRNA Complex(A) Molecular surface of TLR3 dimer (green) with bound dsRNA (Liu et al., 2008)(3CIY). The interaction of the C-terminal capping motifs stabilizes the TLR3dimer.(B) Top view.

Structure

Review

alpha helices on the convex side of the horseshoe and two large,

conserved loops that protrude from the lateral and convex faces

of LRRs 12 and 20, respectively. TLR3-ECD is heavily glycosy-

lated, with 15 predicted N-glycosylation sites, of which 11 are

visible in the crystal structure. The glycans decorate all faces

of the TLR3-ECD, except for the lateral surface on the C-terminal

side of the b sheet. This face offers a large, planar surface for

interaction with dsRNA (Figure 2). In the crystal structure of the

TLR3-ECD-dsRNA complex, the glycan-free surfaces of two

TLR3-ECDs sandwich the dsRNA molecule, generating an

m-shaped structure (Liu et al., 2008) (Figure 5A). No conforma-

tional change in the TLR3-ECD occurs on ligand binding. In the

complex, the dsRNA interacts at two sites on each TLR3-ECD,

one near the N terminus (encompassing LRR-NT and LRRs

1-3), and one near the C terminus (involving LRRs 19–23)

(Figure 5B). In addition, the two ECDs interact with each other

at their LRR-CT motifs. Mutational analyses (Wang et al., 2010)

have established that the simultaneous interaction of all three

sites is required for stable binding of dsRNA to TLR3.

Although the N- and C-terminal dsRNA sites are separated by

55–60 A in each ECD, the two N-terminal sites in the complex are

separated by 110 A. This latter distance correlates with a dsRNA

length of �45 base pairs and explains why dsRNA oligonucleo-

tides less than �40 bp cannot bind or activate TLR3 (Leonard

Stru

et al., 2008). Because cells normally contain short (%25 bp)

stretches of dsRNA for example in miRNA and tRNA hairpins,

the inability of TLR3 to bind dsRNA <40 bp most likely provides

an important mechanism for preventing autoreactive responses

against self dsRNA. In binding dsRNA, the TLR3-ECD interacts

only with the ribose-phosphate backbone, accounting for the

lack of RNA sequence specificity in binding. This feature would

prevent the viruses from escaping detection by mutation. All

binding-site residues are located on the glycan-free surface of

the ECD. Of a particular interest is the Asn413 N-linked pauci-

manose moiety that reaches out from the concave surface and

contacts the dsRNA helix, but the relevance of this interaction

is unclear.

The majority of TLR3/dsRNA interactions are hydrophilic,

involve hydrogen bonds and salt bridges, and account for a total

buried area of 1103 A2 (4.4% of the TLR3-ECD surface). Partic-

ularly important are electrostatic interactions between the phos-

phate groups from the dsRNA backbone and the imidazole rings

of four histidine residues, three in the N-terminal site, and one in

the C-terminal site. Mutation of two of these histidines (H39 and

H60 in the N-terminal site) to alanine abolishes binding and

responsiveness to dsRNA, indicating that the salt bridges

formed by these residues are essential for complex formation.

This could explain the pH-dependency of dsRNA binding

because these side chains would be protonated only below

pH 6.5 and able to interact with the RNA phosphates. The struc-

ture of the complex reveals the main reasons for the inability of

TLR3 to interact with dsDNA. The helical structure of dsDNA is

the B form, whereas dsRNA assumes the A form. The B helix

would not be structurally compatible with the two terminal

binding sites on the TLR3-ECD horseshoe. Also, there are

several hydrogen bonds between TLR3-ECD and the 20-hydroxylgroups of dsRNA that would be missing in dsDNA.

The only interaction between the two TLR3 molecules in the

complex occurs at the interface between the two LRR-CTmotifs,

and therefore this site is responsible for dimerization. In this site,

the two LRR-CTs are related by two-fold symmetry and the inter-

actions between the LRR-CTs are mainly hydrophilic, consisting

of hydrogen bonds and salt bridges. The dimerization site is

essential for dsRNA binding (Wang et al., 2010) because it

correctly positions the four dsRNA binding sites in the complex,

but in addition it also brings the two C-terminal residues within

�25 A of each other. In the cell, two LRR-CT motifs that interact

on the luminal side of an endosome would presumably bring the

two TIR domains together on the cytoplasmic side, forming

a dimeric scaffold on which adaptor molecules could bind and

initiate signaling.

TLR4 and MD-2Lipopolysaccharide (LPS), an essential component of the outer

membrane of Gram-negative bacteria, induces a powerful

inflammatory response that can lead to septic shock and death

(Beutler and Rietschel, 2003). LPS signals through TLR4 by com-

plexing coreceptor MD-2, which is anchored by several

hydrogen bonds to the lateral and concave surface of TLR4-

ECD and contacts residues from the LRR2-LRR10 area

(Kim et al., 2007) (Figure 6A). LPS-binding protein (LBP) and

CD14 deliver and load the LPS to the TLR4-bound MD-2.

cture 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved 453

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

Structure

Review

One of the important factors that determine the inflammatory

potential of an LPS molecule (Rietschel et al., 1994) is the

number of lipid chains in the Lipid A portion of LPS. Six chains

provide an optimal inflammatory activity, whereas Lipid A with

five chains has �100-fold less activity. Ligands with only four

lipid chains, such as lipid IVa and Eritoran, have antagonistic

activity (Teghanemt et al., 2005). Because the lipids interact

with the MD-2 pocket through hydrophobic contacts, variations

in the lipid chain structure can be accommodated by shifting the

position of the chains in the pocket. With a smaller number of

lipid chains, Lipid A can move deeper into the pocket and the

phosphate groups cannot participate in ionic interactions with

TLRs (Park et al., 2009). These two phosphate groups are also

essential for the endotoxic activity of LPS, and deleting either

of them reduces the endotoxic activity by �100-fold (Rietschel

et al., 1994).

TLR4-ECD has 21 LRRs capped by LRR-NT and LRR-CT

motifs. The structures of the TLR4-ECD and several chimeric

454 Structure 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved

fragments of TLR4 capped with hagfish

VLR domains (Kim et al., 2007) show

a horseshoe structure that is much less

planar than the TLR3-ECD. Three subdo-

mains can be differentiated, N-terminal,

central, and C-terminal, with different

degrees of twist and curvature. The

central subdomain contains only one

variable residue between the last leucine

residue of a preceding LRR motif and

the first leucine residue of the next LRR

motif, in contrast with the standard two

variable residues (Figure 1A). The length

of these LRR modules also varies

between 20–30 residues, conferring

a smaller radius and greater twist angle

to the subdomain. The central subdo-

mains of human and mouse TLR4 differ

most in structure, apparently due to the

MD-2 binding to the mTLR4 LRR9 loop.

MD-2 is a coreceptor molecule that

binds both the extracellular domain of

TLR4 and the hydrophobic portion of

LPS (Visintin et al., 2006). The crystal

structure of hMD-2 complexed to lipid

IVa has been determined (Ohto et al.,

2007) (Figure 6D). Lipid IVa, a precursor

of Lipid A, is an antagonist for hTLR4 but an agonist for mTLR4

(Means et al., 2000). The molecule adopts the b cup topology

of the ML family of lipid-binding proteins that have sandwiched

antiparallel b sheets and conserved disulfide bridges (Dere-

wenda et al., 2002). The b sheets of hMD-2 form a very deep

and narrow hydrophobic pocket, with a surface area of �1000

A that can bury the four lipid strands of lipid IVa. The opening

of the pocket is lined by positively charged residues and three

disulfide bridges stabilize the cup-like structure. Based on this

structure, it seemed unlikely that the hMD-2 pocket would be

able to accommodatemore than four lipid chains without confor-

mational changes, but another structure with bound LPS

showed that additional room for the ligand is generated, not by

conformational change, but by displacing the LPS glucosamine

backbone upward by �5 A (Park et al., 2009). In addition to the

displacement, the glucosamine backbones are also rotated by

�180�, interchanging the two phosphate groups. The crystal

structure of the TLR4-ECD/MD-2/Eritoran complex was also

Structure

Review

reported (Kim et al., 2007) (Figure 6E). Eritoran is a synthetic

antagonist with four lipid chains that occupy �90% of the

hMD-2 hydrophobic pocket, whereas the di-glucosamine sugars

are fully exposed to solvent and the phosphate groups form

essential ionic bonds with positively charged residues at the

opening of the pocket. Binding of Eritoran does not induce signif-

icant structural changes in hMD-2. The available structures

suggest that theMD-2 pocket evolved to bind large and structur-

ally different ligands (Kim et al., 2007). Agonists and antagonists

will bind in a similar fashion, but with their glucosamine back-

bones rotated by �180�.The 3.1 A resolution structure of the TLR4/MD-2/LPS complex

(Park et al., 2009) shows two Escherichia coli LPS molecules

bound (Figure 6A). Five of the LPS lipid chains are buried in the

large hydrophobic MD-2 pocket, whereas the sixth one is

exposed and makes hydrophobic contacts with conserved

phenylalanines on the other TLR4-ECD (Figure 6B). The LPS

phosphate groups form ionic interactions with positively charged

residues on MD-2 and TLR4-ECD (Figure 6C). In addition, the

F126 and L87 loops of MD-2 interact with the second TLR4-

ECD molecule. All these interactions bring the two copies of

the TLR4-ECD/MD-2/LPS complex together into a typical

m-shaped signaling complex.

Cells that express TLR4/MD-2 also express a homologous

complex, RP105/MD-1, that regulates the LPS response

(Akashi-Takamura and Miyake, 2008; Divanovic et al., 2007).

RP105 is a type I receptor with an ECD resembling that of

TLR4. However, RP105 lacks a C-terminal TIR domain. MD-1,

like MD-2, binds LPS and a crystal structure of a lipid IVa/

MD-1 complex has been reported (Yoon et al., 2010). Although

both MD-1 and MD-2 are members of the ML family of lipid

binding proteins, they use different surfaces for ligand recogni-

tion. RP105/MD-1 binds directly to TLR4/MD-2, and on B cells

RP105/MD-1 enhances the LPS response (Miyake et al., 1994,

1998; Miura et al., 1998), whereas on macrophages it attenuates

the response (Divanovic et al., 2005).

TLR5TLR5 is one of the few TLRs that recognize a protein PAMP,

bacterial flagellin (Hayashi et al., 2001). It is highly expressed in

gut, especially in lamina propria dendritic cells (Uematsu and

Akira, 2009) where it controls the composition of the microbiota

(Vijay-Kumar et al., 2010).With no structure available, TLR5-ECD

is predicted to contain 20 LRRs, with five loops extending from

the ascending or convex surfaces (Bell et al., 2003). Mutational

analyses of flagellin have located the site recognized by TLR5

as lying in the conserved D1 domain (Donnelly and Steiner,

2002). This domain is exposed in monomeric flagellin, but is

buried in the polymerized flagellin fiber, suggesting that TLR5

recognizes flagellin monomers that are released on depolymer-

ization of flagellin polymers (Smith et al., 2003). The portion of

the TLR5-ECD that interacts with flagellin is less well defined.

Human and mouse TLR5 differ in their ability to recognize WT

or mutated flagellin molecules from different sources (Ander-

sen-Nissen et al., 2007; Miao et al., 2007). Moreover, mouse

TLR5 gains human specificity when one residue, Pro268, is

mutated to its human counterpart, alanine and vice versa.

Sequence analysis predicts that Pro268 lies on the ascending/

convex surface of LRR9, an atypical LRR motif (Bell et al.,

Stru

2003). Thus it is likely that LRR9 plays an important role in

flagellin recognition by TLR5.

The TLR 7, 8, and 9 familyNo structure has yet been reported for any member of the TLR 7,

8, and 9 subfamily. Like TLR3, these TLRs are located in endo-

somes and recognize nucleic acid PAMPs. However, their amino

acid sequences suggest that the structures of TLRs 7-9 ECDs

are markedly different from TLR3 (Bell et al., 2003). TLRs 7–9

ECDs each comprise 25 LRRs and are heavily glycosylated.

They all contain large insertions in LRRs 2, 5, and 8 that most

likely give rise to structures that loop out from the dimerization

surfaces of the ECDs. In addition, the ECDs of TLRs 7–9 contain

stretches of �40 residues between LRRs 14 and 15 that have

undefined structure. Because these stretches are the only

portions that show a high degree of species variability in each

of the three paralogs, it is likely that these regions are relatively

unstructured. TLR9, which recognizes single stranded DNA

with unmethylated CpG sequences, especially in viral and bacte-

rial DNA, has been the most extensively studied paralog of this

family (Hemmi et al., 2000; Kumar et al., 2009). There is evidence

(Latz et al., 2007) that TLR9 undergoes an extensive conforma-

tional change when binding CpG DNA, which would make it

very different from TLR3. A ligand-induced conformational

change could be dependent on the unstructured region if it

served as a hinge within the horseshoe, thus allowing a large

conformational transition to occur on ligand binding. More

recently, several studies have suggested that proteases are

required for TLR9 function (Asagiri et al., 2008; Ewald et al.,

2008; Matsumoto et al., 2008; Park et al., 2008), and that TLR9

is cleaved within the undefined region between LRRs 14 and

15 (Park et al., 2008), consistent with this being an unstructured,

exposed stretch of amino acid residues. Another report suggests

that the C-terminal fragment, lacking the first 14 LRRs and the

undefined region, is active by itself implying that the N-terminal

fragment is not needed for ligand recognition (Park et al.,

2008). This observation seems to be inconsistent with a more

recent study (Peter et al., 2009) showing that mutations within

the N-terminal fragment inactivate TLR9. Indeed it seems

unlikely that a large portion of the TLR9-ECD would be highly

conserved throughout vertebrate evolution, and yet not be

essential for TLR9 function. Clearly, a detailed structural analysis

of the TLR9-ECD is needed to clarify the mechanism of ligand

binding and activation of TLR9 as well as TLRs 7 and 8.

Common Features and Differences betweenthe Signaling ComplexesThe signaling complex structures of the TLRs with their respec-

tive ligands reveal the diverse mechanisms of recognition of

a wide variety of PAMPs. These PAMPs carry characteristic

molecular signatures that are unique to different classes of path-

ogens and are detected by the TLRs. In all structures, the

signaling complex consists of an m-shaped TLR dimer, in which

the ECDN-termini extend to the opposite ends and the C-termini

interact in the middle. Formation of the homo- or heterodimer

supports the hypothesis that dimerization of the ECDs positions

the cytoplasmic TIR domains close enough to dimerize and

initiate a downstream signaling cascade (Jin et al., 2007; Kang

et al., 2009; Liu et al., 2008; Park et al., 2009). In most cases,

cture 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved 455

Figure 7. TLR3 Signaling Complex and Signaling Pathways(A) Structural model of the full-length TLR3/dsRNA signaling complex. Themodel is based on the mTLR3/dsRNA structure (3CIY) and a TLR3 TIR domainhomology model based on the TLR10 TIR structure (2J67). The trans-membrane portions have been modeled as a helices.(B) TLR signaling pathways. TLR3 signals exclusively through TRIF, whereasTLR4 can use both the TRIF and the MyD88 pathways. All other TLRs use theMyD88 pathway.

Structure

Review

two ECDs cradle a single ligand molecule, except for TLR4

where two MD-2 molecules each bind a ligand. Most known

TLR structures do not use the concave beta-sheet surface for

ligand binding, a characteristic mode of binding for other LRR

proteins. TLR4 is an exception because it binds its adaptor

protein MD-2 in the concave surface.

In all TLR complexes, the TLR-ECDs are related by an approx-

imate 2-fold symmetry axis. In the TLR 1, 2, 3, and 6 complexes,

the nonglycosylated surface of two TLR molecules sandwich

a ligand molecule, whereas in the TLR4 complex the coreceptor

molecule MD-2 binds the ligand directly. TLR3 binds its ligand

mainly by hydrogen bonding and electrostatic interactions

whereas hydrophobic interactions dominate the ligand binding

in other known TLR structures. The buried surface area in the

TLR1/TLR2 interaction is similar to that in the TLR3 homodimer.

In the TLR3/dsRNA complex the protein-protein interactions

occur only at the LRR-CT, whereas direct interactions between

TLR1/TLR2 or TLR2/TLR6 occur near the binding pockets. In

the TLR3/dsRNA and TLR4/MD-2/LPS complexes the two

C-terminal residues are �25 A apart, whereas in the TLR1/2

and TLR2/6 complexes they are �40 A apart. This larger

distance may be due to the fact that the native LRR-CT motifs

were replaced with hagfish VLRs, however. In the TLR4/MD-2/

LPS complex, despite the close proximity of the C-termini there

is no direct interaction between the two molecules in this region.

In the known TLR-ligand complexes, ligands bind TLRs by

different mechanisms, but in each case, the ligand bridges two

TLR-ECDs on the same glycan-free surface and forms dimers

with similar overall architecture.Whether this paradigmwill apply

to the TLR7-9 family remains to be determined.

The TIR DomainThe TLRs signal pathogen attack by dimerization of their cyto-

plasmic TIR domains in response to ligand-induced dimerization

of the ectodomains (Figure 7A). TIR dimerization of TLRs is

recognized by TIR domains on the adaptor proteins MyD88,

MAL, TRIF, and TRAM, which then trigger downstream signaling

pathways, leading to the expression of inflammatory cytokines,

various antiviral and antipathogen proteins and to initiation of

the adaptive immune response (Figure 7B). The TIR domain is

one of many evolutionarily conserved building blocks of the

immune system (Figure S1) (Palsson-McDermott and O’Neill,

2007).

The crystal structures of isolated TIR domains have been re-

ported for TLRs 1, 2 (Xu et al., 2000) and 10 (Nyman et al.,

2008) (Table 2), IL-1RAPL (Khan et al., 2004) and for TIR domains

from Arabidopsis thaliana (Chan et al., 2010) and Paracoccus

denitrificans (Chan et al., 2009). In addition, an NMR structure

has been reported for the MyD88 TIR domain (Ohnishi et al.,

2009). In all TIR domains, alternating b strands and a helices

are arranged as a central five-stranded parallel b sheet sur-

rounded by five a helices.

The TIR domains from both TLR1 and TLR2 exist as mono-

mers in the crystal. Despite sharing 50% sequence identity there

are important conformational differences between the two struc-

tures. By contrast, the 2.2 A structure for the TIR domain of

human TLR10 revealed a 2-fold symmetric dimer that has been

taken to represent the signaling dimer for the TIRs (Figure 8)

(Nyman et al., 2008). The BB-loop that joins strands bB and aB

456 Structure 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserve

is one of the essential points of interaction in the dimeric inter-

face, which also contains residues from the DD-loop, and the

aC-helix. In addition to being involved in TIR dimer formation,

the BB-loop is sufficiently exposed to interact with adaptor

proteins during signal transduction. In TLR4, the P681H poly-

morphism, which lies in the BB-loop, abolishes signaling in

response to LPS (Poltorak et al., 1998), indicating that the BB

loop plays an essential role in TLR signaling. The structures of

the plant (Chan et al., 2010) and bacterial TIR domains (Chan

et al., 2009) are similar to their mammalian counterparts.

d

Figure 8. Structure of the TIR Domain Dimer from TLR10(A) Ribbon diagram of the dimeric TIR10, with the two interacting BB-loopshighlighted in gold and red (Nyman et al., 2008) (2J67).(B) Molecular surface of dimeric TIR10.

Structure

Review

However, in the latter a dimer is formed that is very different from

the TLR10 dimer. The BB loops are not involved in dimerization

contacts but are highly exposed on the surface. An important

question left in TLR structural biology is the orientation of TIR

domains that activate the signaling cascade. Available TIR

domain structures lack the region immediately following their

transmembrane (TM) segment, making it harder to predict their

exact orientation. In the TLR10 TIR dimer, the N-termini of both

molecules have to simultaneously point toward the membrane,

to accommodate the linker between the TM segment and the

first N-terminal residue from the TIR.

Purified TIR domains from the MAL and MyD88 adaptor

proteins have been shown to form stable heterodimers in solu-

tion (Dunne et al., 2003). Dunne et al. (2003) also produced

models of the TIR domains from human TLR4, MAL, and

MyD88 and used these to model the interactions between pairs

of these TIR domains. In these models, the BB loop does not

seem to be involved in either TIR dimerization or in heterotypic

interactions with TIRs from the adaptor proteins, which is

contrary to expectation. Clearly, more structures of interacting

TIR domains will be required to better understand how TIR

domains function. In addition to its TIR domain, MyD88 contains

a death domain. After binding to TLR TIR domains, MyD88 death

domains can interact with the death domains of members of the

IRAK family of Ser/Thr kinases, triggering their activation and

initiatingdownstreamsignaling cascades. A structure of a ternary

complex formed by the death domains of humanMyD88, IRAK4,

Stru

and IRAK2 (Lin et al., 2010) showed that these molecules form

a left handed helical structure containing in order 6 MyD88,

4 IRAK4, and 4 IRAK2 death domains. The MyD88-IRAK4 oligo-

meric assembly—also called the myddosome—was also

confirmed by cryo EM and small angle X-ray scattering experi-

ments (Motshwene et al., 2009). There are three types of interac-

tions observed between the death domains in the myddosome.

SNPs in the human MyD88 death domain, S34Y and R98C inter-

fere with the myddosome assembly and may drastically

contribute to susceptibility to infection (George et al., 2011). If

each TLR-TIR dimer binds two MyD88 TIR domains then the

large myddosome superhelix could possibly bridge several acti-

vated receptor dimers into a network (Gay et al., 2011). These

scaffolds might include more than one type of MyD88-depen-

dent TLR, enabling synergistic responses to microbial stimuli.

There are other cases where death domains assemble in super-

helical oligomeric signaling complexes like the death-inducing

signaling complex (Scott et al., 2009) or the PIDDosome

(Park et al., 2007).

ConclusionsTLRs are germ-line encoded pattern recognition receptors that

initiate defensive responses against a wide variety of pathogens.

TLRs are type I membrane receptors with mainly planar, horse-

shoe-shaped LRR ECDs. TLR-ECD-ligand complex structures

are known for TLR1/TLR2 and TLR6/TLR2 with lipopeptides,

TLR3 with dsRNA, and TLR4/MD-2 with LPS. Although the

TLR-ligand interactions are very different, they all produce an

m-shaped dimeric complex with C termini in the middle, and

N-termini on the outside. Pairing of the ECD C-termini can lead

to dimerization of the cytoplasmic TIR domains, which is recog-

nized homotypically by adaptor molecule TIR domains. MyD88,

the most commonly used adaptor, also has death domains that

interact with IRAK kinase death domains to form a superhelical

myddosome, which presumably initiates the signaling cascade.

Despite substantial progress in our understanding of the struc-

tural basis of TLR recognition and signaling, several questions

need to be addressed. There is still no structural data for TLRs

5, 7, 8, and 9. Amino acid sequences of the TLR 7-9 family

suggest that they may differ from other TLRs in both structure

and mode of ligand recognition. How TLR-TIR domains interact

with each other or with TIRs from adaptor molecules is still

unclear. Structures of adaptor molecules would provide insight

into how they translate TIR recognition into IRAK activation.

Better understanding of the structural basis for PAMP recogni-

tion and signaling by TLRs could lead to the development of

adjuvants that specifically bind to TLR-ECDs and activate

TLRs or of anti-inflammatory drugs that block TLR mediated

signaling.

SUPPLEMENTAL INFORMATION

Supplemental Information includes one figure and can be found with thisarticle online at doi:10.1016/j.str.2011.02.004.

ACKNOWLEDGMENTS

This work was supported by the Intramural Research Program of the NationalInstitutes of Health (NIDDK and NCI) and by a National Institutes of Health/Food and Drug Administration intramural biodefense award from NIAID.

cture 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved 457

Structure

Review

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