The LRRs within most of TLRs are flanked by two cysteine clusters, each of which contains two to five cysteine residues (Table 2 and Figures 5, 6, 7, 8, 9, and 10). Here the cysteine clusters on the N- and C-terminal sides of LRRs are termed LRRNT and LRRCT, respectively [58]. The N-terminal cluster usually consists of two cysteines, Cx_5–14C, but sometimes 3, 4 or 5 cysteines. With high frequency, as noted, the last cysteine of the clusters occupies a structurally equivalent position to those of leucines in the HCS part of LRR1. The Cx₈C motif in TLR3 and the Cx₁₀C motif of TLR4 form a disulfide bond [52,59], as does the Cx₁₂C motif in GPIbα [41]. The Cx_5–14C motifs presumably form disulfide bonds. The C-terminal clusters, excepting those in three TLRs (Table 2), contain four cysteines consisting of CxCx_22–25Cx_15–20C. The spacing between the first and the second cysteine that are contained in the last LRR is the same for all the families. The other spacing appears to characterize each family. The CxCx₂₅Cx₁₈C motif in TLR3 forms two disulfide bonds between the first and the third cysteines, and between the second and the fourth cysteines [52]. Such pairs of disulfide bonds have been observed for the CxCx₂₀Cx₂₁C motif in Nogo receptor [38,39] and the CxCx₂₀Cx₁₉C motif in Slit [49]. The disulfide bond connectivity can be inferred for TLRs. The C-terminal cluster for primate TLR4 (CxCx₂₃Cx₁₇C) is different from that of other mammalian TLR4 (CxCx₂₃Cx₁₈C). Only in rainbow trout TLR5 and Takifugu rubripes TLRS5 having no TIR domain, does the C-terminal cluster consists of two cysteines. There are no N-terminal cysteine clusters in TLR1, TLR6, TLR10, TLR15, and dog TLR4. However, the N-terminal amino acid sequence flanking the LRR domain might form a capping structure.

The present analyses of LRRs within vertebrate TLRs indicate that there are at least two types of LRR motifs; "typical"; "T", LRR, LxxLxLxxNxLxxLxxxxFxxLxx and "bacterial"; "S", LRR, LxxLxLxxNxLxxLPx(x)LPxx. Vertebrate TLRs contain 16–28 LRRs (Table 2 and Figures 5, 6, 7, 8, 9, and 10). Bell et al., [60] have proposed that the ECDs of human TLRs comprised 19–25 LRRs including both "T" and "S" LRRs. Each member of human TLRs contain 1–2 times less LRRs than those identified here. Furthermore, in the TLR1 family (TLR1, TLR2, TLR6 and TLR10) the LRRs at the central parts are aligned differently to each other. Such a difference is also seen in TLR4, TLR5, and TLR7. The alignments of TLR3, TLR8 and TLR9 are nearly identical except the first LRR at the N-terminus of the LRR domain and the last LRR at its C-terminus.

The TLR7 family (TLR7, TLR8, TLR9 and green puffer TLR) have 27 LRRs and an additional 58–73 residues at the end of LRR15 (Figures 9, and 10). Such a long region is also observed in chicken TLR15 (Figure 10). Gibbard et al., [61] have considered two horseshoe domains of LRRs for human TLR8. That is, LRR15 has been separated into an LRR motif and 40 residues of undetermined structure. Most of the known LRR structures have a cap, which shields the hydrophobic core at the N- and C-terminii of LRRs. We suggest that these 40 residues function as the cap of the horseshoe structure, an intervening of hydrophobic core of LRR with a specific feature in TLRs. Thus, it can be concluded that the LRRs in vertebrate TLRs form one or two distinct horseshoe structures. Future structure determinations should resolve the question.

The TLR1 family (TLR1, TLR2, TLR6, and TLR10) and the TLR4 family share a common feature, the central part of the LRR domain has a more irregular motif compared with those at the N- and C-terminal parts. The LRR structure in the three families of TLR1, TLR4 and TLR7 might show a structural flexibility at the central part. Alternatively, the central part would play a key role in the function.

The LRR arc structures can be characterized by three parameters- the inner radius of the arc (R), the mean rotation angle about the central axis relating one β-strand to the next (ϕ¯ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacuaHvpGAgaqeaaaa@2E90@), and the tilt angle of the parallel β-strand direction per turn (θ_t). A 3D circle fitting method to calculate these geometrical parameters has been developed [55]. The TLR3-LRR arc yields R = 26.5–26.6Å, ϕ¯ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacuaHvpGAgaqeaaaa@2E90@ = 10.8–10.9° and θ_t = 24.5–26.7°. The TLR3-LRR belongs to "typical" type. This R value is comparable to 22–36 Å for the LRR arcs in Slit, FSHr, nogo receptor, decorin, and GPIbα with "typical" LRRs [8,55,58]. In contrast, the θ_t value is comparable to only those for Slit (-21°) and FSHr (-40°). Also the θ_t value corresponds to 19–40° for ribonuclease inhibitor and 15° for tropomodulin with "RI-like" LRRs. That is, the TLR3-LRR arc is nearly flat. This indicates that all other TLRs except for the TLR7 family and TLR15 might adopt flat LRR arc.

The present analysis reveals that the TLR7 family consisting of TLR7, TLR8 and TLR9 and green puffer TLR contains the super-motif consisting of STT. Such super motifs have been observed in various LRR proteins [8,11]. One of them is the SLRP family. The SLRP family forms five distinct subfamilies. Class I consists of biglycan, decorin, and asporin. Class II has three subclasses: lumican plus fibromodulin (IIA), PRELP plus keratocan (IIB), and osteoadherin (IIC). Class III consists of epiphycan, osteoglycin and opticin. Class IV is more distantly related and consists of chondroadherin and nyctalopin. Class V consists of podocan. Their classes except for class IV contain the super-motif. Super-motifs, S and T, similar to those in SLRP are also present in asporin-like proteins from human and mouse, mouse fibromodulin-like proteins, biglycan-like proteins from sea lamprey, oligodendrocyte-myelin and glycoprotein (OMGP), the FLRT family from human, mouse and Xenopus, and human ECM2 [8,62]. Furthermore, a preliminary analysis indicates that nephrocan, a novel member of the SLRP family [63], contains an STT motif. These observations suggest strongly that "bacterial" and "typical" LRRs evolved from a common precursor.

A number of amino acid polymorphisms, which occur in LRRs, have been reported in TLRs. Arbour et al., [64] first identified two mutations of human TLR4, D299G and T399I, which were associated with diminished airway responsiveness to inhaled LPS. Since then, these two mutations have been studied for their association with various infectious and inflammatory diseases; results regarding the effects of these mutations have been inconclusive [65-71]. D299G and T399I occur in LRR11 and LRR15, respectively (Figure 7). D299G is near the convex part, while T399I is located on the loop C-terminal to the convex part. Very recently, Ohara et al., [72] reported that one mutation, T135A, was associated with poorly-differentiated gastric adenocarcinomas. T135A in LRR5 occur at position 9 in the HCS part (Figure 7). Such a mutation has been observed in many LRR proteins such as nyctalopin, keratocan, GPIbα, GPIbβ and GPIX, which are associated with human diseases [58]. Position 9 is generally occupied by Asn or Cy and sometimes by Thr or Ser, whose side chains form hydrogen bonds in the loop structure [58]. The T135A mutation may disrupt the hydrogen bond pattern in the loop.

Mouse TLR9 plays a role in defense against systemic mouse cytomegalovirus infection. Mice with the mutation, L499P, are highly susceptible to mouse cytomegalovirus infection and shows low levels of cytokine induction and natural killer activation on viral infection [73]. L499P is located at the short loop that connects the helical structure on the convex part (in LRR17) and the β-strand on concave part (in LRR18) (Figure 10). That is, L499P in LRR18 occur at position 1 in the HCS part. The side chain of L499 is completely buried in the LRR arc. Such a mutation is also observed in trk-A and nyctalopin, which are associated with human diseases [58]. The mutation of D543A in human TLR8 abolishes the binding of CpG DNA [61]. D543A in LRR19 occur at position 1 in the VS part. Thus, D543A is located at the edge between the convex and the concave parts of the LRR arc. The Cys-to-Ala mutations in the VS part of LRR9 (C257A, C260A, C267A, and C270A) completely abolish signaling by TLR8 [61].

Hidaka et al., [74] detected one mutation, F303, in human TLR3 in one of three patients with influenza-associated encephalopathy. This was a loss-of-function mutation. F303S in LRR12 is located at position 4 in the HCS part. The side chain of F303 is completely buried in the LRR arc. Two mutations, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions [75]. These two mutations occur in LRR21.

The structures of twenty-two different LRR proteins have been determined. They are ribonuclease inhibitor (RI) [2NBH, I1DJ, LA4Y, 1Z7X], GTPase-activating protein (RanGAP) [1YRG, 1K5D, 1K5G], tropomodulin (Tmod) [1IO0, 1PGV], S-phase kinase-associated protein 2 (Skp2) [1FQV], YopM [1G9U], four internalins, Inl-B [1D0B], Inl-H [1H6U], Inl-A [106T, 106V, 106S] and Inl-C [1XEU], spliceosomal U2A' protein [1A9N], mRNA export factor (TAP) [1FT8, 1F01], rab geranylgeranyltransferase α-subunit (RabGGTα) [1DCE, 1LTX], Chlamydomonas outer arm dynein light chain 1 (DLC-1) [1DS9], polygalacturonase-inhibiting protein (PGIP) [10GQ], nogo receptor/nogo-66 receptor (NgR) [10ZN, 1P9A], glycoprotein Ibα (GPIbα) [1M0Z, 1GWB, 1QYY, 1M10, 1SQ0, 1P8V, 100K, 1P9A, 1U0N], decorin [1XCD, 1XKU, 1XEC], biglycan [2FT3], Slit [1W8A], CD14 [1WWL], follicle-stimulating hormone receptor (FSHr) [1XWD], TLR3 [1ZIW, 2A0Z], and human lingo-1 [2ID5].

The LRRs alignments within the TLR family were made for TLR1 from four species (human [Q15399, Q5FWG5, Q6FI64, Q32MK3], mouse [Q9EPQ1], pig [Q4LDR7, Q59HI9], Takifugu rubripes [Q5H727]); TLR2 from 17 species (human [O60603], mouse [Q9QUN7, Q8K3D9, Q811T5], pig [Q59HI8, Q5DX20, Q76L24], chicken [Q9DD78 (TLR2.1), Q9DGB6 (TLR2.2)], bovine [Q95LA9], rat [Q6YGU2], dog [Q689D1], rabbit [AAM50059], goat [ABI31733], horse [AAR08196], hamster [Q9R1F8], Cynomolgus monkey [Q95M53], domestic water buffalo [Q2PZH4], Nilgai [Q2V897], Takifugu rubripes [Q5H725], zebrafish [Q6TS42], Japanese flounder [Q76CT8]); TLR3 from 9 species (human [O15455, Q4VAL2, Q504W0], mouse [Q99MB1, Q3TM31, Q499F3], bovine [Q5TJ58, Q5TJ59], rat [Q7TNI8], buffalo [Q1G1A3], Rhesus macaque [Q3BBY1], Takifugu rubripes [Q5H721], zebrafish [Q6IWL5, Q32PW5], Japanese flounder [Q76CT7, Q76CT9]; TLR4 from 17 species (human [O00206, Q5VZI7, Q5VZI8, Q5VZI9], mouse [Q9QUK6, Q5RGT4, Q8K2T5], pig [Q68Y56, Q2TNK4, Q5F4K7, Q401C7], bovine [Q9GL65, Q6WCD5, Q8SQ55], rat [Q9QX05], hamster [Q9WV82], cat [P58727], lowland gorilla [Q8SPE8], horse [Q9MYW3], Pygmy chimpanzee [Q9TTN0], olive baboon [Q9TSP2], orangutan [Q8SPE9], Nilgai [Q2V898], American bison [Q3ZD70], dog [Q8SQH3], rabbit [AAM50060]; zebrafish [Q6NV08, Q6TS41(TLR4b)]; TLR5 from 8 species (human [O60602], pig [Q59HI7], mouse [Q9JLF7], bovine [Q2LDA0], chicken [Q4ZJ82], Japanese house mouse [Q1ZZX0], Takifugu rubripes [Q5H720, Q5H716(TLRS5)], rainbow trout [Q7ZT81]); TLR6 from 5 species (human [Q9Y2C9], mouse [Q9EPW9, Q7TPC5], rat [Q6P690], pig [Q59HI6, Q76L23], bovine [Q704V6, Q706D2]; TLR7 from 4 species (human [Q9NYK1], mouse [P58681, Q548J0], dog [Q2L4T3], Takifugu rubripes [Q5H719]); TLR8 from 4 species ((human [Q9NR97, Q495P4, Q495P6, Q495P7], mouse [P58682], pig [Q865R7], Takifugu rubripes [Q5H718]); TLR9 from 12 species (human [Q9NR96[, mouse [Q9EQU3], pig [Q5I2M3, Q865R8], bovine [Q5I2M5, Q866B2], dog [Q5I2M8], cat [Q5I2M7], Japanese flounder [Q2ABQ3], horse [Q2EEY0], sheep Q5I2M4], Ma's night monkey [Q56R09], Gilthead sea bream [Q3L273, Q3L274], Takifugu rubripes [Q5H717]]; TLR10 from two species (human [Q9BXR5, Q5FWG4, Q32MI7, Q32MI8], pig [Q4LDR6, Q59HI5]); TLR11 from mouse [Q6R5P0, Q32ME8]; TLR12 from mouse [Q6QNU9]; TLR13 from mouse [Q6R5N8]; TLR14 from Takifugu rubripes [Q5H726] and zebrafish [XP_687315]; TLR15 from chicken [ABB71177], TLR21 from Takifugu rubripes [NP_001027751], TLR22 from Takifugu rubripes [Q5H723], TLR23 from Takifugu rubripes [AAW70378], and TLR from rainbow trout [Q6KCC7, Q4LBC9], Atlantic salmon [Q2A132], goldfish [Q801F9]), Japanese lamprey [Q33E92, Q33E93] and green puffer (Fragment) [Q4S0D3]).

The protein secondary structure prediction by SSpro4.0 [13,76,77]and Proteus [12,78] were utilized for the determination and assignments of LRRs within TLRs. Signal peptide prediction was performed by SignalP 3.0 [79,80]. The prediction of membrane-spanning regions in proteins was performed by the TMHMM Program [81,82]. The PFAM program [83] was used to detect LRRs in TLRs.

Multiple sequence alignments and sequence similarity searches were performed at Bioinformatic Center, Insitute for Chemical Research, Kyoto University [84]. Dot-matrix comparisons were performed using the Blosum90 scoring matrix. The program was made in house. Window sizes and stringencies are indicated in figure legends.