the structural biology of toll-like receptors · structure review the structural biology of...
TRANSCRIPT
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: [email protected] 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
REFERENCES
Akashi-Takamura, S., and Miyake, K. (2008). TLR accessory molecules. Curr.Opin. Immunol. 20, 420–425.
Andersen-Nissen, E., Smith, K.D., Bonneau, R., Strong, R.K., and Aderem, A.(2007). A conserved surface on Toll-like receptor 5 recognizes bacterialflagellin. J. Exp. Med. 204, 393–403.
Asagiri, M., Hirai, T., Kunigami, T., Kamano, S., Gober, H.J., Okamoto, K.,Nishikawa, K., Latz, E., Golenbock, D.T., Aoki, K., et al. (2008). Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis.Science 319, 624–627.
Bell, J.K., Mullen, G.E., Leifer, C.A., Mazzoni, A., Davies, D.R., and Segal, D.M.(2003). Leucine-rich repeats and pathogen recognition in Toll-like receptors.Trends Immunol. 24, 528–533.
Bell, J.K., Botos, I., Hall, P.R., Askins, J., Shiloach, J., Segal, D.M., and Davies,D.R. (2005). The molecular structure of the Toll-like receptor 3 ligand-bindingdomain. Proc. Natl. Acad. Sci. USA 102, 10976–10980.
Bella, J., Hindle, K.L., McEwan, P.A., and Lovell, S.C. (2008). The leucine-richrepeat structure. Cell. Mol. Life Sci. 65, 2307–2333.
Beutler, B., andRietschel, E.T. (2003). Innate immune sensing and its roots: thestory of endotoxin. Nat. Rev. Immunol. 3, 169–176.
Brinkmann, M.M., Spooner, E., Hoebe, K., Beutler, B., Ploegh, H.L., and Kim,Y.M. (2007). The interaction between the ER membrane protein UNC93B andTLR3, 7, and 9 is crucial for TLR signaling. J. Cell Biol. 177, 265–275.
Burch-Smith, T.M., and Dinesh-Kumar, S.P. (2007). The functions of plant TIRdomains. Sci. STKE 2007, e46.
Chan, S.L., Low, L.Y., Hsu, S., Li, S., Liu, T., Santelli, E., Le, N.G., Reed, J.C.,Woods, V.L., Jr., and Pascual, J. (2009). Molecular mimicry in innate immunity:crystal structure of a bacterial TIR domain. J. Biol. Chem. 284, 21386–21392.
Chan, S.L., Mukasa, T., Santelli, E., Low, L.Y., and Pascual, J. (2010). Thecrystal structure of a TIR domain fromArabidopsis thaliana reveals a conservedhelical region unique to plants. Protein Sci. 19, 155–161.
Choe, J., Kelker, M.S., and Wilson, I.A. (2005). Crystal structure of human toll-like receptor 3 (TLR3) ectodomain. Science 309, 581–585.
DeLano,W.L. (2002). The PyMOLMolecular Graphics System (SanCarlos, CA:DeLano Scientific).
Deng, L., Velikovsky, C.A., Xu, G., Iyer, L.M., Tasumi, S., Kerzic, M.C., Flajnik,M.F., Aravind, L., Pancer, Z., and Mariuzza, R.A. (2010). A structural basis forantigen recognition by the T cell-like lymphocytes of sea lamprey. Proc. Natl.Acad. Sci. USA 107, 13408–13413.
Derewenda, U., Li, J., Derewenda, Z., Dauter, Z., Mueller, G.A., Rule, G.S., andBenjamin, D.C. (2002). The crystal structure of a major dust mite allergen Derp 2, and its biological implications. J. Mol. Biol. 318, 189–197.
Divanovic, S., Trompette, A., Atabani, S.F., Madan, R., Golenbock, D.T., Visin-tin, A., Finberg, R.W., Tarakhovsky, A., Vogel, S.N., Belkaid, Y., et al. (2005).Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptorhomolog RP105. Nat. Immunol. 6, 571–578.
Divanovic, S., Trompette, A., Petiniot, L.K., Allen, J.L., Flick, L.M., Belkaid, Y.,Madan, R., Haky, J.J., and Karp, C.L. (2007). Regulation of TLR4 signaling andthe host interface with pathogens and danger: the role of RP105. J. Leukoc.Biol. 82, 265–271.
Donnelly, M.A., and Steiner, T.S. (2002). Two nonadjacent regions in enteroag-gregative Escherichia coli flagellin are required for activation of toll-likereceptor 5. J. Biol. Chem. 277, 40456–40461.
Dunne, A., Ejdeback, M., Ludidi, P.L., O’Neill, L.A., andGay, N.J. (2003). Struc-tural complementarity of Toll/interleukin-1 receptor domains in Toll-like recep-tors and the adaptors Mal and MyD88. J. Biol. Chem. 278, 41443–41451.
Erridge, C. (2010). Endogenous ligands of TLR2 and TLR4: agonists or assis-tants? J. Leukoc. Biol. 87, 989–999.
Ewald, S.E., Lee, B.L., Lau, L., Wickliffe, K.E., Shi, G.P., Chapman, H.A., andBarton, G.M. (2008). The ectodomain of Toll-like receptor 9 is cleaved togenerate a functional receptor. Nature 456, 658–662.
458 Structure 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserve
Gay, N.J., andGangloff, M. (2007). Structure and function of Toll receptors andtheir ligands. Annu. Rev. Biochem. 76, 141–165.
Gay, N.J., Gangloff, M., and O’Neill, L.A. (2011). What the Myddosome struc-ture tells us about the initiation of innate immunity. Trends Immunol. 32,104–109.
George, J., Motshwene, P.G., Wang, H., Kubarenko, A.V., Rautanen, A., Mills,T.C., Hill, A.V., Gay, N.J., andWeber, A.N. (2011). Two humanMYD88 variants,S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly. J. Biol.Chem. 286, 1341–1353.
Govindaraj, R.G., Manavalan, B., Lee, G., and Choi, S. (2010). Molecularmodeling-based evaluation of hTLR10 and identification of potential ligandsin Toll-like receptor signaling. PLoS ONE 5, e12713.
Han, S.H., Kim, J.H., Martin, M., Michalek, S.M., and Nahm, M.H. (2003).Pneumococcal lipoteichoic acid (LTA) is not as potent as staphylococcalLTA in stimulating Toll-like receptor 2. Infect. Immun. 71, 5541–5548.
Han, B.W., Herrin, B.R., Cooper, M.D., and Wilson, I.A. (2008). Antigen recog-nition by variable lymphocyte receptors. Science 321, 1834–1837.
Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R., Yi, E.C., Goodlett, D.R., Eng,J.K., Akira, S., Underhill, D.M., and Aderem, A. (2001). The innate immuneresponse to bacterial flagellin is mediated by Toll-like receptor 5. Nature410, 1099–1103.
He, X.L., Bazan, J.F., McDermott, G., Park, J.B., Wang, K., Tessier-Lavigne,M., He, Z., and Garcia, K.C. (2003). Structure of the Nogo receptor ectodo-main: a recognition module implicated in myelin inhibition. Neuron 38,177–185.
Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H.,Matsumoto,M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000). A Toll-likereceptor recognizes bacterial DNA. Nature 408, 740–745.
Huizinga, E.G., Tsuji, S., Romijn, R.A., Schiphorst, M.E., de Groot, P.G., Sixma,J.J., and Gros, P. (2002). Structures of glycoprotein Ibalpha and its complexwith von Willebrand factor A1 domain. Science 297, 1176–1179.
Janeway, C.A., Jr., and Medzhitov, R. (2002). Innate immune recognition.Annu. Rev. Immunol. 20, 197–216.
Jin, M.S., Kim, S.E., Heo, J.Y., Lee, M.E., Kim, H.M., Paik, S.G., Lee, H., andLee, J.O. (2007). Crystal structure of the TLR1-TLR2 heterodimer induced bybinding of a tri-acylated lipopeptide. Cell 130, 1071–1082.
Jung, D., and Alt, F.W. (2004). Unraveling V(D)J recombination; insights intogene regulation. Cell 116, 299–311.
Kajava, A.V. (1998). Structural diversity of leucine-rich repeat proteins. J. Mol.Biol. 277, 519–527.
Kang, J.Y., Nan, X., Jin, M.S., Youn, S.J., Ryu, Y.H., Mah, S., Han, S.H., Lee,H., Paik, S.G., and Lee, J.O. (2009). Recognition of lipopeptide patterns byToll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 31, 873–884.
Khan, J.A., Brint, E.K., O’Neill, L.A., and Tong, L. (2004). Crystal structure of theToll/interleukin-1 receptor domain of human IL-1RAPL. J. Biol. Chem. 279,31664–31670.
Kim, H.M., Park, B.S., Kim, J.I., Kim, S.E., Lee, J., Oh, S.C., Enkhbayar, P.,Matsushima, N., Lee, H., Yoo, O.J., and Lee, J.O. (2007). Crystal structure ofthe TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell130, 906–917.
Kim, Y.M., Brinkmann,M.M., Paquet, M.E., and Ploegh, H.L. (2008). UNC93B1delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452,234–238.
Kobe, B., and Deisenhofer, J. (1995). A structural basis of the interactionsbetween leucine-rich repeats and protein ligands. Nature 374, 183–186.
Kumar, H., Kawai, T., and Akira, S. (2009). Pathogen recognition in the innateimmune response. Biochem. J. 420, 1–16.
Latz, E., Verma, A., Visintin, A., Gong, M., Sirois, C.M., Klein, D.C., Monks,B.G., McKnight, C.J., Lamphier, M.S., Duprex, W.P., et al. (2007). Ligand-induced conformational changes allosterically activate Toll-like receptor 9.Nat. Immunol. 8, 772–779.
d
Structure
Review
Leonard, J.N., Ghirlando, R., Askins, J., Bell, J.K., Margulies, D.H., Davies,D.R., and Segal, D.M. (2008). The TLR3 signaling complex forms by coopera-tive receptor dimerization. Proc. Natl. Acad. Sci. USA 105, 258–263.
Lin, S.C., Lo, Y.C., and Wu, H. (2010). Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465, 885–890.
Liu, L., Botos, I., Wang, Y., Leonard, J.N., Shiloach, J., Segal, D.M., andDavies, D.R. (2008). Structural basis of toll-like receptor 3 signaling withdouble-stranded RNA. Science 320, 379–381.
Matsumoto, F., Saitoh, S., Fukui, R., Kobayashi, T., Tanimura, N., Konno, K.,Kusumoto, Y., Akashi-Takamura, S., and Miyake, K. (2008). Cathepsins arerequired for Toll-like receptor 9 responses. Biochem. Biophys. Res. Commun.367, 693–699.
Matsushima, N., Tanaka, T., Enkhbayar, P., Mikami, T., Taga, M., Yamada, K.,and Kuroki, Y. (2007). Comparative sequence analysis of leucine-rich repeats(LRRs) within vertebrate toll-like receptors. BMC Genomics 8, 124.
Matzinger, P. (1994). Tolerance, danger, and the extended family. Annu. Rev.Immunol. 12, 991–1045.
Means, T.K., Golenbock, D.T., and Fenton, M.J. (2000). The biology of Toll-likereceptors. Cytokine Growth Factor Rev. 11, 219–232.
Medzhitov, R. (2007). Recognition of microorganisms and activation of theimmune response. Nature 449, 819–826.
Miao, E.A., Andersen-Nissen, E., Warren, S.E., and Aderem, A. (2007). TLR5and Ipaf: dual sensors of bacterial flagellin in the innate immune system.Semin. Immunopathol. 29, 275–288.
Miura, Y., Shimazu, R., Miyake, K., Akashi, S., Ogata, H., Yamashita, Y.,Narisawa, Y., and Kimoto, M. (1998). RP105 is associated with MD-1 andtransmits an activation signal in human B cells. Blood 92, 2815–2822.
Miyake, K., Yamashita, Y., Hitoshi, Y., Takatsu, K., and Kimoto, M. (1994).Murine B cell proliferation and protection from apoptosis with an antibodyagainst a 105-kD molecule: unresponsiveness of X-linked immunodeficientB cells. J. Exp. Med. 180, 1217–1224.
Miyake, K., Shimazu, R., Kondo, J., Niki, T., Akashi, S., Ogata, H., Yamashita,Y., Miura, Y., and Kimoto, M. (1998). MouseMD-1, amolecule that is physicallyassociated with RP105 and positively regulates its expression. J. Immunol.161, 1348–1353.
Motshwene, P.G., Moncrieffe, M.C., Grossmann, J.G., Kao, C., Ayaluru, M.,Sandercock, A.M., Robinson, C.V., Latz, E., and Gay, N.J. (2009). An oligo-meric signaling platform formed by the Toll-like receptor signal transducersMyD88 and IRAK-4. J. Biol. Chem. 284, 25404–25411.
Nyman, T., Stenmark, P., Flodin, S., Johansson, I., Hammarstrom, M., andNordlund, P. (2008). The crystal structure of the human toll-like receptor 10cytoplasmic domain reveals a putative signaling dimer. J. Biol. Chem. 283,11861–11865.
O’Neill, L.A., and Bowie, A.G. (2007). The family of five: TIR-domain-containingadaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364.
Ohnishi, H., Tochio, H., Kato, Z., Orii, K.E., Li, A., Kimura, T., Hiroaki, H.,Kondo, N., and Shirakawa, M. (2009). Structural basis for the multiple interac-tions of the MyD88 TIR domain in TLR4 signaling. Proc. Natl. Acad. Sci. USA106, 10260–10265.
Ohto, U., Fukase, K., Miyake, K., and Satow, Y. (2007). Crystal structures ofhuman MD-2 and its complex with antiendotoxic lipid IVa. Science 316,1632–1634.
Omueti, K.O.,Mazur, D.J., Thompson,K.S., Lyle, E.A., andTapping, R.I. (2007).The polymorphism P315L of human toll-like receptor 1 impairs innate immunesensing of microbial cell wall components. J. Immunol. 178, 6387–6394.
Palsson-McDermott, E.M., and O’Neill, L.A. (2007). Building an immunesystem from nine domains. Biochem. Soc. Trans. 35, 1437–1444.
Park, H.H., Logette, E., Raunser, S., Cuenin, S., Walz, T., Tschopp, J., andWu,H. (2007). Death domain assembly mechanism revealed by crystal structure ofthe oligomeric PIDDosome core complex. Cell 128, 533–546.
Park, B., Brinkmann,M.M., Spooner, E., Lee, C.C., Kim, Y.M., and Ploegh, H.L.(2008). Proteolytic cleavage in an endolysosomal compartment is required foractivation of Toll-like receptor 9. Nat. Immunol. 9, 1407–1414.
Stru
Park, B.S., Song, D.H., Kim, H.M., Choi, B.S., Lee, H., and Lee, J.O. (2009). Thestructural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex.Nature 458, 1191–1195.
Peter, M.E., Kubarenko, A.V., Weber, A.N., and Dalpke, A.H. (2009). Identifica-tion of an N-terminal recognition site in TLR9 that contributes to CpG-DNA-mediated receptor activation. J. Immunol. 182, 7690–7697.
Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van, H.C., Du, X., Birdwell, D.,Alejos, E., Silva, M., Galanos, C., et al. (1998). Defective LPS signaling inC3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282,2085–2088.
Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow,H., Ulmer, A.J., Zahringer, U., Seydel, U., Di, P.F., et al. (1994). Bacterial endo-toxin: molecular relationships of structure to activity and function. FASEB J. 8,217–225.
Roach, J.C., Glusman, G., Rowen, L., Kaur, A., Purcell, M.K., Smith, K.D.,Hood, L.E., and Aderem, A. (2005). The evolution of vertebrate Toll-like recep-tors. Proc. Natl. Acad. Sci. USA 102, 9577–9582.
Schatz, D.G., and Spanopoulou, E. (2005). Biochemistry of V(D)J recombina-tion. Curr. Top. Microbiol. Immunol. 290, 49–85.
Scott, F.L., Stec, B., Pop, C., Dobaczewska, M.K., Lee, J.J., Monosov, E.,Robinson, H., Salvesen, G.S., Schwarzenbacher, R., and Riedl, S.J. (2009).The Fas-FADD death domain complex structure unravels signalling byreceptor clustering. Nature 457, 1019–1022.
Smith, K.D., Andersen-Nissen, E., Hayashi, F., Strobe, K., Bergman, M.A.,Barrett, S.L., Cookson, B.T., and Aderem, A. (2003). Toll-like receptor 5 recog-nizes a conserved site on flagellin required for protofilament formation andbacterial motility. Nat. Immunol. 4, 1247–1253.
Takeda, K., Kaisho, T., and Akira, S. (2003). Toll-like receptors. Annu. Rev.Immunol. 21, 335–376.
Tao, X., Xu, Y., Zheng, Y., Beg, A.A., and Tong, L. (2002). An extensively asso-ciated dimer in the structure of the C713S mutant of the TIR domain of humanTLR2. Biochem. Biophys. Res. Commun. 299, 216–221.
Teghanemt, A., Zhang, D., Levis, E.N., Weiss, J.P., and Gioannini, T.L. (2005).Molecular basis of reduced potency of underacylated endotoxins. J. Immunol.175, 4669–4676.
Uematsu, S., and Akira, S. (2009). Immune responses of TLR5(+) lamina prop-ria dendritic cells in enterobacterial infection. J. Gastroenterol. 44, 803–811.
Uff, S., Clemetson, J.M., Harrison, T., Clemetson, K.J., and Emsley, J. (2002).Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domainreveals an unmasking mechanism for receptor activation. J. Biol. Chem.277, 35657–35663.
Velikovsky, C.A., Deng, L., Tasumi, S., Iyer, L.M., Kerzic, M.C., Aravind, L.,Pancer, Z., and Mariuzza, R.A. (2009). Structure of a lamprey variable lympho-cyte receptor in complex with a protein antigen. Nat. Struct. Mol. Biol. 16,725–730.
Vijay-Kumar, M., Aitken, J.D., Carvalho, F.A., Cullender, T.C., Mwangi, S.,Srinivasan, S., Sitaraman, S.V., Knight, R., Ley, R.E., and Gewirtz, A.T.(2010). Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 328, 228–231.
Visintin, A., Iliev, D.B., Monks, B.G., Halmen, K.A., andGolenbock, D.T. (2006).MD-2. Immunobiology 211, 437–447.
Wang, Y., Liu, L., Davies, D.R., and Segal, D.M. (2010). Dimerization of Toll-likereceptor 3 (TLR3) is required for ligand binding. J. Biol. Chem. 285, 36836–36841.
Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J.L., and Tong, L.(2000). Structural basis for signal transduction by the Toll/interleukin-1receptor domains. Nature 408, 111–115.
Yang, D., Tewary, P.,, de la Rosa, G., Wei, F., and Oppenheim, J.J. (2010). Thealarmin functions of high-mobility group proteins. Biochim. Biophys. Acta1799, 157–163.
Yoon, S.I., Hong, M., Han, G.W., and Wilson, I.A. (2010). Crystal structure ofsoluble MD-1 and its interaction with lipid IVa. Proc. Natl. Acad. Sci. USA107, 10990–10995.
cture 19, April 13, 2011 ª2011 Elsevier Ltd All rights reserved 459