Identification of an N-Terminal Recognition Site in TLR9 That Contributes to CpG-DNA-Mediated Activation

This information is current as Mirjam E. Peter, Andriy V. Kubarenko, Alexander N. R. of September 29, 2021. Weber and Alexander H. Dalpke J Immunol 2009; 182:7690-7697; ; doi: 10.4049/jimmunol.0900819 http://www.jimmunol.org/content/182/12/7690 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification of an N-Terminal Recognition Site in TLR9 That Contributes to CpG-DNA-Mediated Receptor Activation1

Mirjam E. Peter,2* Andriy V. Kubarenko,2† Alexander N. R. Weber,3† and Alexander H. Dalpke3*

Although it is well established that TLR9 recognizes CpG-DNA, the structural details of ligand-receptor interaction are still mostly unknown. The extracellular domain of TLR9 is composed of 25 leucine-rich repeat (LRR) motifs, 5 of which bear inserting sequences that do not conform to the LRR consensus motif. In this study, we show that the functional integrity of the extracellular domain of murine TLR9 is lost by deletion of individual LRR motifs. When deleting only the inserting sequences, we observed that LRR2, 5, and 8 contribute to receptor activation by CpG-DNA. The latter deletions did not affect receptor dimerization but inhibited CpG-DNA binding. On the basis of a homology modeling approach, we furthermore identify a positively charged region in the N terminus that is essential for CpG-DNA-induced TLR9 activation. This interaction site mirrors findings previously shown

for the structural recognition of dsRNA by TLR3 and hints toward a general principle of nucleic acid recognition by the respective Downloaded from TLR. The Journal of Immunology, 2009, 182: 7690–7697.

nnate immunity relies on TLR to detect invading microor- sented to the receptor through a binding , MD-2 (6). In ganisms (1). TLR are germline-encoded type I integral mem- contrast, the crystal structure of TLR3 bound to dsRNA showed I brane whose extracellular domain (ECD)4 is two binding sites for RNA that are formed by charged patches on responsible for ligand binding. The ECD of TLR is composed of its surface (7–9). One is located in the N-terminal part of the ECD http://www.jimmunol.org/ 19–25 leucine-rich repeats (LRR) (2). Each LRR forms a loop in (involving the LRR-NT and LRR1–3), and the other one is located which conserved hydrophobic residues point inward, and several in the C terminus with the irregular LRR20 contributing to RNA of these loops build-up a horseshoe-shaped ECD, which is N- and binding. Mutational studies confirmed the binding of RNA to res- C-terminally flanked by so-called cysteine flanking regions, idues in a region encompassing LRR20 and deletion of the inser- termed LRR-NT and -CT, respectively. The first 10 aa of LRR are tion in LRR20 led to a complete loss of function (10). conserved in all LRR subtypes and form a ␤-sheet shaping the In contrast to other TLR, the nucleic acid recognizing TLR3, 7, concave surface of the ECD, whereas the remaining portion of the 8, and 9 recognize their ligands in intracellular compartments such LRR is variable among the different subtypes and forms the con- as endosomes. A further noticeable feature of TLR7–9 is that the vex surface. All TLR contain varying numbers of “irregular” LRR irregular LRR of these receptors are located at the same positions by guest on September 29, 2021 that do not entirely conform to the consensus motif but bear in- and that the insertions are very homologous (3). Another similarity serting stretches of amino acids, which protrude from the horse- between these receptors is the presence of a less structured region shoe-shaped backbone and have been proposed to be involved in between LRR14 and 15 with low similarity to the LRR consensus. ligand binding (3). It has been proposed that this region may bring flexibility to the To date, three modes of ligand binding by mammalian TLR receptor (11). In contrast to TLR3, which recognizes dsRNA se- have been identified (4). In the TLR1/TLR2 heterodimer the acyl quence independently (12), TLR7–9 are stimulated by nucleic ac- chains of the Pam3CSK4 ligand are directly inserted into hydro- ids in a sequence-specific manner (13, 14), which implies that in phobic channels stretching LRR9–12 (5). For TLR4, LPS is pre- addition to the general binding characteristics of TLR3 for nucleic acids, recognition by TLR7–9 may be more complex. Surprisingly, for TLR9, it has been recently shown that cleavage of the ECD *Department of Medical Microbiology and Hygiene, University Heidelberg, Heidel- occurs and it was proposed that the C-terminal fragment starting berg, Germany; and †Junior Research Group “Toll-like Receptors and Cancer,” Ger- man Cancer Research Center, Heidelberg, Germany from LRR15 mediates ligand recognition (15, 16). Received for publication March 12, 2009. Accepted for publication April 10, 2009. Whereas receptor dimerization and subsequent signal transduc- tion occur upon ligand binding for TLR2-TLR1 and TLR3, it was The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance published for TLR9 that this receptor exists as a preformed dimer with 18 U.S.C. Section 1734 solely to indicate this fact. (11). Binding of stimulatory DNA was proposed to result in a 1 This study was supported by the German Research Foundation (Deutsche For- conformational change in the receptor, which decreases the diam- schungsgemeinschaft DA592/3 (to A.H.D.); Emmy Noether program (to A.N.R.W.)) eter of the ECD. This process was suggested to bring the TIR and by the German Cancer Research Center (to A.V.K., A.N.R.W.). domains in close proximity, thereby activating downstream sig- 2 M.E.P. and A.V.K. contributed equally. naling. In this study, we tested the hypothesis that insertion-bear- 3 Address correspondence and reprint requests to Dr. Alexander H. Dalpke, De- partment of Medical Microbiology and Hygiene, Hygiene-Institute, University ing LRRs within TLR9 contribute to ligand recognition. Addition- Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany; E-mail ad- ally, we sought to determine whether specific binding sites in the dress: [email protected] or Dr. Alexander N. R. Weber, TLR9 ECD can be mapped. Junior Group “Toll-like Receptors and Cancer,” German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. E-mail address: [email protected] Materials and Methods 4 Abbreviations used in this paper: ECD, extracellular domain; HA, hemagglutinin; Cells and reagents LRR, leucine-rich repeat; ODN, oligodeoxynucleotide; WT, wild type. CpG-oligodeoxynucleotide (ODN) no. 1668 (TCCATGACGTTCCTGA Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 TGCT) was custom synthesized by MWG-Biotech either 3Ј-biotinylated or www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900819 The Journal of Immunology 7691

unmodified. Resiquimod (R848) was purchased from InvivoGen. LPS from various TLR ligands. After 24 h of stimulation, TNF-␣ was determined in Salmonella minnesota was provided by U. Seydel (Research Center Bor- cell-free supernatants by ELISA, according to the manufacturer’s instruc- stel, Borstel, Germany). DMEM, RPMI 1640 medium, and FCS were pur- tion, and fresh media were added to the cells. Other 24 h later supernatants chased from Biochrom. Abs were received from Cell Signaling Technol- were analyzed for NO accumulation photometrically (550 nm) by mixing ogy. Immobilized protein A was obtained from Thermo Scientific, equal parts of supernatant and Griess reagent (1:1 mixture of 1% sulfanil-

streptavidin-agarose was from Prozyme, and the cathepsin inhibitor z-FA- amide/5% H3PO4 and 0.1% naphthyl-ethylenediamine dihydrochloride). fmk was from Biovision. RAW 264.7 cells, a murine macrophage cell line, were a gift from R. Schumann (Institute for Microbiology and Hygiene, Homology modeling and structure analysis Berlin, Germany). HEK293 cells were obtained from S. Bauer (Institute for Immunology, Marburg, Germany). Homology modeling of mouse TLR9 ECD was conducted as previously described (18) based on the published human TLR3 ECD crystal struc- Site-directed mutagenesis tures (7, 19). In brief, the TLR9 ECD was generated stepwise by mod- eling N- and C-terminal subdomains and LRR14 individually using the mTLR9-HA-plasmid was constructed by replacing the YFP-tag in TLR9- MODELLER package (20). These were manually assembled, and spa- YFP (obtained by T. Espevik, Institute of Cancer Research and Molecular tial violations resulting from the manual docking procedure were cor- Medicine, Trondheim, Norway) with an HA-tag, using the restriction en- rected using GROMACS molecular dynamics (21). The complete ECD Ј zymes XhoI and Bsp1407I and custom synthesized 5 -phosphorylated model was then scored for energy and sterical correctness using the ODN encoding the hemagglutinin (HA)-tag sequence YPYDVPDYA. The ANOLEA (22), VERIFY_3D and ERRAT (http://nihserver.mbi.ucla. same approach was used to obtain a TLR9-myc-plasmid (encoded se- edu/) online servers. Structure analysis was conducted in SwissPBD quence: EEQKLISEEDL). Site-directed mutagenesis was conducted using Viewer (23) and PyMol (www.pymol.org) visualization software using primers containing the desired mutations using the QuikChange from PDB2PQR (24), PropKa (25), and APBS (26) packages for charge sur- Stratagene. Sequences of the primers can be made available upon request. face calculation. Several clones were subjected to automated sequencing to confirm the Downloaded from mutation and to exclude further undesired mutations. Results Reporter experiments The aim of this study was to identify regions within the ECD of For reporter gene experiments a firefly luciferase reporter construct with a TLR9 involved in receptor activation. We first tested the hy- 6ϫ NF-␬B responsive element was used. HEK293 cells (0.2 ϫ 106) were pothesis that irregular LRRs bearing inserting sequences that do transfected in 24-well format and a volume of 500 ␮l. mTLR9-HA (25 ng) not conform to the consensus LRR motif might play a role in or the indicated mutant plasmid or 100 ng of mTLR7 plasmid (obtained

receptor activation. In murine TLR9 such sequences can be http://www.jimmunol.org/ from S. Bauer) together with 25 ng of NF-␬B-reporter plasmid encoding Firefly luciferase and 100 ng pRL-TK (Promega) encoding Renilla lucif- found in LRR2, 5, 8, and 11 (insertion at position 10, number- erase were transfected using Lipofectamine 2000 (Invitrogen). Forty-eight ing according to Ref. 3) and in LRR20 (after position 15) as hours after transfection, cells were stimulated, and luciferase activities shown in Fig. 1A. were determined an additional 6 h later using the Dual Luciferase Reporter Assay System Kit (Promega). Shown are mean values of duplicates of one Deletion of single LRR modules abolishes TLR9 activity of at least three independent experiments. Initially, we made single deletions of each irregular LRR and Western blotting tested the mutants (termed Del_LRRx, x denotes number of HEK293 cells were transfected in a 6-well format with 4 ␮gofthe LRR; Table I) in an NF-␬B-dependent reporter assay in indicated plasmid. Forty-eight hours later, cells were lysed for 30 min HEK293 cells. Deletion of any complete LRR bearing the in- by guest on September 29, 2021 on ice in 250 ␮l of lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Igepal, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM serting sequences was not tolerated and led to loss of function PMSF, 1 ␮g/ml each of aprotinin, leupeptin, and pepstatin, 1 mM of the receptor (Fig. 1B). Only in the case of LRR11, some

Na3VO4, and 1 mM NaF). Lysates were cleared by centrifugation at 4°C residual activity remained. Surprisingly, even the deletion of for 15 min at 11,000 ϫ g. Equal amounts of lysates were fractionated LRR16, an entirely regular LRR, which was included as a con- by 8% SDS-PAGE and electrotransferred to polyvinylidene difluoride trol, resulted in loss of receptor activity. We concluded that this membranes. The membranes were blocked with TBS (pH 7.8)/5% non- fat dry milk/0.05% Tween 20 and were blotted with the indicated abs approach influences the overall structure of the horseshoe shape according to the manufacturer’s protocol. HRP-labeled Abs were visu- of the ECD, thus interfering with receptor activity irrespective alized by ECL (Amersham Biosciences). of the position of the LRR. Pull-down assay On the basis of the high homology between TLR9 and TLR7 with regard to the position and composition of the irregular LRR, HEK293 cells were transfected with TLR9-HA wild type (WT) or the we next switched LRR2, 5, 8, 11, and 20 from TLR9 to those from indicated mutant plasmid and lysed as described above. Lysates were in- cubated with 5 ␮M3Ј-biotin-CpG-ODN at 4°C for 1.5 h. Subsequently, 20 TLR7 (sLRRx; Table I). Again, all of the mutants featuring only ␮l of streptavidin-agarose beads was added for 60 min. Beads were washed one switched LRR module showed loss of responsiveness toward five times in lysis buffer, and eluted were used for Western blot the TLR9 stimulus CpG-ODN (Fig. 1C). Corroborating the above analysis (17). HRP-labeled Abs were visualized by ECL (Amersham Bio- findings, even the switch of an LRR without insertion (sLRR16) or sciences) using the Chemi-Smart 2000 video documentation system from PEQLAB Biotechnologie in the automated mode to avoid overexposure switching a highly homologous LRR (sLRR6; see also below) led and saturation of the signal. Quantification of the detected signals was done to loss of responsiveness. This suggests an intricate spatial ar- using the Bio-1D Advanced-Software (PEQLAB Biotechnologie). The rangement of LRR in the TLR9 ECD whose disruption results in amount of precipitated TLR9 was normalized to the amount of receptor in the loss of receptor function. Not surprisingly, chimeras where all the lysate and compared with the TLR9 WT pull-down. LRR containing irregular insertions of TLR9 were replaced with Immunoprecipitation the corresponding ones of TLR7 (LRR2, 5, 8, 11, and 20) also A total of 1 ϫ 106 HEK293 cells was cotransfected with 4 ␮g of TLR9- showed loss of CpG-ODN responsiveness (data not shown). More- myc and 4 ␮g of TLR9-HA WT or the indicated mutant in a volume of 1 over, the latter mutant did not become responsive to stimulation ml medium. A total of 200 ␮l of the lysates of these cells was incubated with the TLR7 ligand R848 (data not shown). Thus, it was not with 3 ␮l of anti-myc Ab for3hat4°C. After further incubation with 20 possible to switch ligand specificity by exchanging the irregular ␮ l of protein A beads for 2 h, precipitates were washed five times, and LRR modules between TLR7 and TLR9, which contrasts find- eluted proteins were used for Western blot analysis. ings for TLR1 and TLR6, where switching of different LRR Determination of TNF-␣ and NO secretion modules led to an exchange in ligand specificity of these two A total of 1.5 ϫ 105 RAW264.7 cells was preincubated with the indicated receptors (27). The correct expression of all above mutant con- amount of cathepsin inhibitor z-FA-FMK for 2 h and then stimulated with structs was confirmed by Western blotting and flow cytometry 7692 TLR9 N TERMINUS CONTRIBUTES TO CpG-DNA RECOGNITION

Deletion of single LRR modules does not affect TLR9 dimer formation In contrast to TLR3, TLR9 is present as preformed dimers (11). To analyze whether the above mutants could still form such dimers, we conducted coimmunoprecipitations of two differently tagged TLR9s. No stimulation was performed, as preformed dimers were assumed to exist. Deletion of any complete LRR in the region between LRR-NT and LRR20 had no impact on the formation of preformed dimers (Fig. 1D). Coimmunoprecipitations were spe- cific as TLR4-HA was not detected in precipitates from cells co- transfected with both TLR4-HA and TLR9-myc. The results were in concordance with data from TLR3 where only regions in the very C-terminal part of the ECD participated in direct protein- protein interactions (LRR22, 23, and LRR-CT) (9). When detect- ing TLR9 by Western blotting, we noted that in addition to the full-length protein a second protein of ϳ95 kDa was detected. In accordance with two recent reports on cleavage of TLR9 (15, 16), this band would correspond to an N-terminally truncated TLR9. Of note, none of the mutations appeared to lead to a loss of the cleav- Downloaded from age product.

N-terminally located irregular LRR contribute to CpG-ODN binding and TLR9 activation

To examine only the influence of the insertions in the irregular http://www.jimmunol.org/ LRR and in order not to disturb the structure by changing the number of LRR modules in the ECD, we generated mutants which lacked only the amino acids that did not conform to the consensus LRR-motif. Removal of insertions in the N-terminal LRR2, 5, and 8 (Del_Insx; Table I) led to a complete loss in receptor function (Fig. 2A). In contrast, deleting the insertions in LRR11 and LRR20 did not (Del_Ins11) or only partially (Del_Ins20) inhibit TLR9 activation, indicating that these in-

sertions are not involved in ligand recognition and receptor ac- by guest on September 29, 2021 tivation and that in general deletion of individual insertions may be tolerated. Correct expression of the mutant proteins was ver- ified by Western blotting, FACS analysis, and confocal micros- copy (Fig. 2D and data not shown). FIGURE 1. Deletion or exchange of complete LRR in TLR9 abolishes receptor function. A, Homology model of the TLR9 ectodomain with irregular Additionally, we analyzed whether this set of mutants also ex- LRR2, 5, 8, 11, 20 highlighted in red and regular LRR6, 16 in blue. B and C, erted a dominant-negative effect when coexpressed together with HEK293 cells were transfected with the indicated TLR9 mutant plasmids, TLR9 WT and stimulated with CpG-ODN (Fig. 2B). Deletion of stimulated with 1 ␮M CpG-ODN 48 h later, and analyzed for NF-␬B reporter the insertions in LRR2, 5, and 8 but not in LRR11 and 20 exhibited gene activity (mean Ϯ SD, one of four experiments). D, Dimer formation was a dominant-negative effect when coexpressed with TLR9 WT. assessed by immunoprecipitation with myc Ab and subsequent analysis with HA- Whereas Del_Ins20 showed no changes in NF-␬B-activation co- -possible cleavage product of TLR9). transfection of Del_Ins11 with TLR9 WT even increased the ac ,ء) tag-specific Abs by Western blot analysis tivation, comparable to cotransfection of empty vector with TLR9 of YFP-tagged mutants (Fig. 1D and data not shown). All tested WT. Thus, Del_Ins11 behaved similar to TLR9 WT. mutants were expressed at comparable levels and at the ex- We next sought to address whether the mutants were still able to pected size (130 kDa). bind to CpG-ODN. Therefore, HEK293 cells were transfected with

Table I. Generated constructs of TLR9

Names of Deleted Amino Acids Names of Amino Acids of TLR7 Inserted in Deletion Mutant in TLR9 WT Switch Mutant the Corresponding Del_LRRX Mutant

Del_LRR2 88–123 sLRR2 90–127 Del_LRR5 168–199 sLRR5 173–204 Del_LRR8 245–284 sLRR8 250–290 Del_LRR11 335–364 sLRR11 341–370 Del_LRR16 498–522 Del_LRR20 600–629 sLRR20 621–651 Del_Ins2 98–110 sLRR6 200–220 (TLR9) replaced by 205–225 (TLR7) Del_Ins5 178–185 sLRR6_1 200–209 (TLR9) replaced by 205–214 (TLR7) Del_Ins8 255–270 sLRR6_2 210–220 (TLR9) replaced by 215–225 (TLR7) Del_Ins11 345–349 Del_Ins20 615–620 The Journal of Immunology 7693

FIGURE 2. Removal or modification of LRR insertions interferes with TLR9 activity. HEK293 cells were transfected with the indicated TLR9 plasmids either alone (A and C) or together with untagged TLR9 WT (1:50 ratio) (B) or myc-TLR9 WT (D). Forty-eight-hour posttransfection cells were stimulated, and NF-␬B-dependent luciferase activity was measured 6 h poststimulation (A and B). C, Binding of biotinylated CpG-ODN to HA-tagged TLR9 was determined by streptavidin-agarose pull-down and quantitative Western blotting. D, Dimer formation was assessed by immunoprecipitation with myc Ab and subsequent analysis .(possible cleavage product of TLR9; mean Ϯ SD of at least two (D) or three (A–C) independent experiments ,ء) with HA-tag-specific Abs by Western blot analysis

the respective mutants or TLR9 WT and the receptor was precip- are involved in ribose backbone binding. This site is located in one Downloaded from itated from protein lysates using biotinylated CpG-ODN (17). The of two positively charged areas in the TLR3 N terminus (Fig. 4A). amount of precipitated protein was normalized to the amount of We therefore analyzed conserved histidine residues in the protein present in the lysate and compared with precipitated TLR9 WT (Fig. 2C). Of note, this kind of binding assay showed a con- siderable level of unspecific binding (here to TLR4-HA) probably

due to the nature of the phosphothioate modification in CpG-ODN http://www.jimmunol.org/ (28). Therefore, precise image quantification was conducted to en- able a comparative analysis. For mutants lacking the specific in- sertions in the irregular LRR (Del_Insx), only Del_Ins11 and 20 retained the ability to bind CpG-ODN, in line with the activity measured earlier (Fig. 2A). Conversely, the insertions in LRR2, 5, and 8 were involved in ligand binding (Fig. 2C). Interestingly, all mutants lacking insertions were able to engage in dimer formation with TLR9 WT (Fig. 2D) as observed before for the mutants with deletions of whole LRR. These findings indicate that the insertions by guest on September 29, 2021 are not involved in receptor-receptor interaction but are important for ligand recognition.

Surface charge is important for TLR9 activation by CpG-DNA Having established that individual LRR insertions contribute to TLR9 function, we sought to address if distinct patches in the N-terminal part of the TLR9 ECD contribute to receptor func- tion. A three-dimensional homology model of the mTLR9 ectodomain was generated (18). LRR6 is the most conserved LRR between TLR9 and 7 (Fig. 3A). The first 10 residues of this LRR (part 6_1; Fig. 3A) are the ones that have the same con- sensus pattern in all LRR subforms and form the ␤-sheets build- ing the concave surface of the horseshoe-shaped ECD. Replace- ment of these first 10 residues was tolerated (Fig. 3C). Unexpectedly, the remaining 11 aa (part 6_2; Fig. 3A) could not be exchanged, even though only 4 of the 11 residues differ between the two receptors (position TLR9/TLR7: 211L/V, 213K/A, 216R/T, and 217Q/T). However, surface charge was drastically altered when replacing residues in part 2 of TLR9 with those from TLR7 (Fig. 3B). Mutation of only one of the four differing residues did not affect receptor function (Fig. 3C). Only for L211V less absolute activity was measured, FIGURE 3. LRR6 is required for due to a positively but due to a lower control activation, this equaled to an identical charged surface patch. A, Stick representation of LRR6 with part 1 (residues 200– x-fold induction. Changing individual residues in our model did 209) colored in orange and part 2 (residues 210–221) colored green. The sequence not greatly affect surface charge (data not shown). alignment of murine TLR9 and 7 is shown for LRR6. B, Surface charge compu- tation of LRR6 part 2 before change of TLR9 residues 210–221 (WT LRR6; top An N-terminal positively charged area is important for TLR9 panel) and after change to TLR7 residues 214–225 (sLRR6; bottom panel) with activity residues of interest in TLR9 labeled. Red, negative charge; blue, positive charge. C, HEK293 cells were transfected with the indicated TLR9 plasmids, stimulated In TLR3 two binding sites for RNA have been reported (8, 9) and with 1 ␮M CpG-ODN 48 h later, and NF-␬B-dependent luciferase activity was in the N-terminal site conserved histidine residues H39 and H60 determined as before (mean Ϯ SD, one of three experiments). 7694 TLR9 N TERMINUS CONTRIBUTES TO CpG-DNA RECOGNITION

R74, which generate a second charged patch in close proximity to the three histidines and adjacent to the irregular insert in LRR2 (Fig. 4A). Surprisingly, mutations of the histidines had no effect on CpG-induced TLR9 activation (Fig. 4B). In contrast, mutating K51 or R74 to the uncharged amino acid methionine led to nearly com- plete loss of function of the receptor. The glutamate mutant did not have stronger effects in the case of R74, whereas for K51 the residual activity still present in the K51M mutant vanished for K51E. The N-terminal point mutations did not affect expression of TLR9 as all mutants were detected at comparable levels and at the expected size in Western blot analysis (Fig. 4C). As shown before for all other mutants, a second band of lower m.w. occurred, in- dicating that cleavage of the receptor was not influenced by the mutants. However, the point mutants that lacked activity (K51M and R74M) showed abolished CpG-ODN binding, whereas the histidine mutants were still able to interact with CpG-ODN (Fig. 4D). The results confirm that the identified N-terminal patch rep-

resents a ligand interaction site. Downloaded from Decreased activity of TLR9 mutants is not due to impaired TLR9 cleavage Recently, it has been published that the ECD of TLR9 is cleaved within the endosome to generate a functional receptor (15, 16).

Furthermore, it was suggested that the C-terminal cleavage frag- http://www.jimmunol.org/ ment (encompassing LRR15s and further) would be sufficient to mediate receptor activation. Although a different model system was used, such a mode of ligand recognition contrasts with our observation of a specific contribution of both N-terminal insertions (LRR2, 5, and 8) and a specific N-terminal positively charged patch (K51 and R74) to CpG-DNA recognition. Reexamining our protein expression data of C-terminally tagged TLR9-HA mutants we could observe a second band in Western blots of approximately the size published (90 kDa; cf. Figs. 2D and 4C). This band could by guest on September 29, 2021 be observed for all mutants analyzed, so that the loss of function of several of these mutants is unlikely to be due to interference with the cleavage reaction. Moreover, in the coimmunoprecipita- tion studies we performed, the putative C-terminal fragment was also present in the precipitate (Figs. 1D and 2D). Because several point mutations or deletions within the N-terminal part of TLR9 ECD abolished TLR function, our results argue for a role of this part or fragment in receptor activation, even though the ECD is cleaved and that the C-terminal fragment by itself is capable of CpG-ODN-binding (16). FIGURE 4. A positively charged patch around lysine 51 and arginine 74 Cathepsins B and L were shown to be able of cleaving TLR9 in is essential for TLR9 signaling. A, Surface charge computation of the mu- vitro, but inhibitors did not influence TLR9 processing and mice rine TLR3 (left panel) and murine TLR9 (right panel) N termini. Red, deficient of different cathepsins had no defect in TLR9 signaling negative charge; blue, positive charge. Distinct positively charged patches (15). Inhibition of lysosomal cystein proteases using the inhibitor marked by dashed circles and individual residues of interest labeled. B and z-FA-fmk inhibited TLR9 but not TLR4 signaling in a recent re- C, HEK293 cells were transfected with the indicated plasmids. B, TLR9 port (16), whereas a previous publication also observed inhibition function was assessed upon stimulation with CpG-ODN by NF-␬B reporter of LPS signaling due to interference with NF-␬B transactivation gene activity as before. C, TLR9 expression was analyzed by Western (29). We therefore reanalyzed the effects of z-FA-fmk on TLR Blotting. D, Binding of biotinylated CpG-ODN to HA-tagged TLR9 was signaling in RAW264.7 macrophages. We observed that z-FA-fmk determined by streptavidin-agarose pull-down and quantitative Western did not only inhibit TLR7 and TLR9 signaling but also interfered blotting. with LPS-induced NO and TNF-␣ secretion from RAW264.7 cells (Fig. 5, A and B), suggesting that it did not specifically inhibit N-terminal part of TLR9 ECD, which are likely to carry a positive endosomally located TLR. The effects were not due to cytotoxicity charge under the mild acidic pH conditions in the endosome and because MTT assays showed no alterations by inhibitor treatment thus would be charge-complementary to the DNA backbone (17). (data not shown). To further exclude that the loss of function of Three conserved histidine residues, H76, H77, and H79, in K51M and R74M was due to effects on receptor cleavage, we LRR-NT and LRR1 of TLR9 were found to form a positive patch. introduced the same mutations into the chimeric receptor These were mutated to phenylalanine or glutamic acid residues TLR9N4C (30). This receptor is composed of the ECD of TLR9 thereby removing positive or introducing negative charges, respec- and the transmembrane and cytoplasmic domain of TLR4, thereby tively. We also mutated two positively charged residues, K51 and localizing to the cell surface. As reported by others (16), we could The Journal of Immunology 7695

LRR (LRR16). Our data suggest that the correct number of LRR modules within the ECD is essential for proper receptor function, because deletion of any complete LRR tested led to complete loss of responsiveness toward CpG-ODN, irrespective of the position of the LRR and whether it was irregular or not. Taken together, the results of this first approach showed that the spatial arrangement of individual LRR in TLR9 is crucial for proper receptor function. Interestingly, mutants with missing LRR were still able to form preformed dimers but lost the ability to bind CpG-ODN (data not shown). These data sharply contrast published data for TLR2 where it was possible to delete the seven N-terminal LRR without

disturbing the recognition of Pam3CSK4 (31). Thus, in TLR2, the recognition site for lipopeptides was mapped to a distinct region by mutational studies, and these findings were subsequently con- firmed by x-ray crystallography (5). In TLR3, deletion of some (but not all) complete LRR was also shown to render TLR3 un- responsive (32). As even the deletion or switching of typical LRR domains was not tolerated in TLR9, this receptor appears even

more sensitive to such manipulation, and ligand recognition in Downloaded from TLR9 follows different spatial constraints. However, by deleting only the inserted sequences in the irreg- ular LRR, we identified specific LRR (LRR2, 5, and 8) in the N-terminal part of the ECD to be essential for receptor function. In contrast, the lack of the insertions in LRR11 and 20 did not or only

partly interfere with TLR9 activity. Our binding data suggest that http://www.jimmunol.org/ loss of function was due to decreased binding of the respective mutants to CpG-DNA, highlighting the importance of LRRs2, 5, and 8 for CpG-ODN engagement. The inactive mutants were, as expected from the results before, still able to dimerize with TLR9 WT and acted in a dominant- negative manner on TLR9 WT stimulation. This suggests that in TLR9 C-terminal regions (e.g., C-terminal LRR and LRR-CT) en- gage in receptor-receptor interactions similar to TLR3 (9) or Dro- sophila Toll (33, 34), whereas ligand binding necessarily requires by guest on September 29, 2021 the insertions in N-terminal LRR. Our data do not rule out that the FIGURE 5. N-terminal recognition of CpG-DNA is independent of region located between LRR11 and 20 may harbor an additional TLR9 cleavage. A and B, RAW264.7 cells were preincubated with 5, 10, nucleic acid binding site similar to the C-terminal binding site for or 20 ␮M z-FA-FMK for 2 h and then stimulated with 300 nM CpG-ODN, dsRNA by TLR3 (8, 9) (Fig. 6). Indeed, it has been reported that 10 ng/ml LPS, or 300 ng/ml R848. Twenty-four hours or 48 h later, TNF-␣ LRR17 contains a region with sequence similarity to methyl-CpG Ϫ and NO2 , respectively, were determined in the supernatant. C and D, binding proteins (35). D535 and Y537 are located in this region HEK293 cells were transfected with the indicated plasmids. TLR9 function and on the same side of the TLR9 ECD as K51, R74, and the LRR was assessed upon stimulation with CpG-ODN by NF-␬B reporter gene insertions according to our homology model (Fig. 6, A and D). ,ء)(activity (C), and TLR9 expression was analyzed by western blotting (D possible cleavage product of TLR9). Mutation of these residues to alanine was shown to abrogate TLR9 responsiveness to and binding of CpG-ODN (36). We therefore suggest that the region surrounding D535 and Y537 may be the observe no cleavage product of TLR9N4C WT in Western blot functional equivalent of the second binding site for dsRNA in analyses (Fig. 5D). Nevertheless, point mutations in the ECD of TLR3 (residues N515, N517, H539, N541, and R544; Fig. 6) (9). TLR9N4C did affect receptor function in a similar way as observed Furthermore, we could show that the lack of the insertions in for TLR9 WT: K51M and R74M led to complete loss of CpG- LRR2, 5, and 8 led to diminished ligand binding. Thus, in TLR9, induced activation whereas H77F had no influence (Fig. 5C). In these specific insertions are involved in the recognition of ligand. Western blot analyses, the mutant forms of TLR9N4C showed To test whether the irregular LRR contribute to the ligand speci- only a single band, indicating that despite the lack of cleavage of ficity of TLR9 and 7, we substituted the irregular LRR in TLR9 by TLR9N4C, the residues K51 and R74 are critical for receptor ac- the corresponding ones of TLR7. Unexpectedly, even the ex- tivation in this chimeric receptor. Taken together, the results rule change of only one LRR led to loss of responsiveness toward the out an interference with receptor cleavage to be causative for im- TLR9 ligand CpG-ODN. Surprisingly, this was also true for proper function of the mutants K51 and R74. LRR16, which fits the consensus LRR motif perfectly. Moreover, we could not turn the specificity toward R848, even when all ir- Discussion regular LRR from TLR9 were shuffled to TLR7. This again con- To identify regions in TLR9 involved in receptor activation and trasts with data from TLR2 and 6. In these TLR, the only LRR ligand binding, we conducted a mutational approach with a focus insertion is located in LRR11. In mutational studies, Meng et al. on the role of irregular LRR, which contain inserting sequences. In (31) switched regions between human and murine TLR2, showing TLR9, such insertions can be observed in LRR2, 5, 8, and 11 that the species-specific response to trilauroylated lipopeptides was (inserting at position 10) and in LRR20 (after position 15). We first mediated by LRR7–10 of TLR2 and that segments of the TLR2 deleted these insertion-bearing LRR and one additional regular ECD can be modified without the loss of receptor function. In our 7696 TLR9 N TERMINUS CONTRIBUTES TO CpG-DNA RECOGNITION

experiments, exchange of even the most highly homologous LRR6 (or parts thereof) in TLR9 by that of TLR7 resulted in deleterious effects on TLR9 responses. For TLR3, it has been reported that several conserved histidine residues in the N-terminal part of the ECD directly bind to the negatively charged ribose backbone of RNA. It was proposed that this contributes to the pH dependency of RNA binding to TLR3, because in the mildly acidified endosomal compartments, the his- tidine imidazole rings would be protonated and thus charge-com- plementary to the RNA backbone. Our results also suggest a role for the N-terminal part of the ECD in binding of the nucleic acid ligand of TLR9. However, whereas the N-terminal histidines 76, 77, and 79 located in LRR-NT and LRR1 were dispensable for function, we observed that two positively charged amino acids located in close proximity of the histidine residues, namely K51 and R74, were of crucial importance. Substitution of K51 or R74 with neutral amino acids led to complete inactivity or in the case of R74M to a strongly diminished response of TLR9. By intro- ducing a negative charge at position 74, this residual activity was Downloaded from abolished. It is interesting to note that TLR3 also contains two positively charged patches (Fig. 4A). The first one is dispensable for signaling as illustrated by the fact that R64 and R65 can be mutated to glutamic acid without loss of function (8). The second one, however, containing H39 and H60, is involved and ligand binding and mutation to alanine or lysine abrogates signaling (8, http://www.jimmunol.org/ 9). Our data suggest that K51 and R74 are the functional equiva- lent of this TLR3 histidine patch and that the histidines in TLR9 do not play a role in ligand binding, similarly to R64 and R65 in TLR3. This was confirmed by ligand-binding studies showing that these two amino acids were involved in binding of CpG-ODN, thereby showing that surface charges in patch 2 but not patch 1 contribute to receptor activation in TLR9. Structural determinants

of ligand binding may thus be similar in the nucleic acid sensors by guest on September 29, 2021 TLR3 and TLR9 but may depend on different residues. Recently, it was shown that the ECD of TLR9 undergoes cleav- age in the endosome, and it was proposed that this step is indis- pensable for TLR9 function (15, 16). Such a mode would imply that the C-terminal cleavage fragment is sufficient for CpG-DNA binding and receptor activation and would hardly be compatible with our findings that clearly indicate a role of the N terminus of TLR9 for receptor activation. We can show that the putative cleav- age product can be observed for all mutants (but not for the TLR9 constructs located to the cell surface), thus ruling out that the mu- tants show loss of activity due to failure to undergo cleavage. It is possible that these seemingly contrasting sets of data could be reconciled if C-terminal fragment and full-length receptor chains remain strongly associated after cleavage—a feature that was re- cently shown for Drosophila Toll (33)—and that this complex has a higher affinity for CpG-DNA. To clarify this point, it would be FIGURE 6. Nucleic acid recognition in TLR3 and 9 follows similar of interest to compare the reported immunoprecipitation results structural principles. A, Surface representations of the generated TLR9 with binding studies using CpG-ODN and purified TLR9 ectodo- ECD model in side-view (top) and upon 90° rotation (horizontal: bottom main (36), as well as the truncations and point mutations described left, vertical: bottom right). Proposed two binding regions are highlighted here and elsewhere (15, 16). Moreover, in our hands, the cathep- by boxes. Insertions of the LRR2, 5, and 8 contributing to receptor acti- vation are depicted in green colors. Patch 2 residues in the N terminus, sinB/L inhibitor z-FA-fmk did not specifically inhibit signaling ␬ important for signaling, are shown in red, patch 1 residues, dispensable for from endosomally located TLR but affected NF- B activation in signaling, are shown in blue. Putative DNA positioning is marked as pink, response to multiple TLR as observed previously (29). dashed region. B, Analogous recognition of dsRNA by TLR3. Surface We conclude that the N-terminal part of the ECD of TLR9 is representation of complex structure (pdb code: 3ciy) with two binding necessary for proper CpG-DNA-mediated receptor activation as regions highlighted by boxes, poly(I:C) ligand in pink. C and D, Close-up multiple mutations in this region showed loss of function without of the proposed N- and C-terminal regions of TLR9 responsible for CpG- affecting expression, dimerization, or cleavage. We do not exclude ODN binding (36). Residues identified as important for ligand binding and that cleavage of TLR9 occurs at some stage of receptor activation receptor activation are highlighted in red. Important residues are shown in ball-and-stick representation in red. and that a C-terminal fragment may play a distinct role in signal activation or regulation, but our data argue that the N terminus is The Journal of Immunology 7697 necessary for full receptor activation in the HEK293 complemen- 9. Liu, L., I. Botos, Y. Wang, J. N. Leonard, J. Shiloach, D. M. Segal, and tation system. More specifically we suggest that the positively D. R. Davies. 2008. Structural basis of Toll-like receptor 3 signaling with double- stranded RNA. Science 320: 379–381. charged patch 2 in LRR-NT and LRR1, which also is in proximity 10. Bell, J. K., J. Askins, P. R. Hall, D. R. Davies, and D. M. Segal. 2006. The to the insertions of LRR2 and 5 (Fig. 6C), represents a binding site dsRNA binding site of human Toll-like receptor 3. Proc. Natl. Acad. Sci. USA 103: 8792–8797. for CpG-DNA similar to such a site in TLR3. 11. Latz, E., A. Verma, A. Visintin, M. Gong, C. M. Sirois, D. C. Klein, In of the previously reported findings on D535 and Y537, B. G. Monks, C. J. McKnight, M. S. Lamphier, W. P. Duprex, et al. 2007. we thus propose a model for nucleic acid recognition by TLR9 Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat. Immunol. 8: 772–779. (Fig. 6A) similar to the ones put forward recently for TLR3 (8, 9) 12. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition (Fig. 6B), in which the nucleic acid ligand is bound at two distinct of double-stranded RNA and activation of NF-␬B by Toll-like receptor 3. Nature receptor sites. Although it is difficult to envisage how such a mode 413: 732–738. 13. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, of binding could be used by small-molecule agonists such as R848, G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger we propose that nucleic acid recognition by TLR7 and 8 may fol- direct activation. Nature 374: 546–549. 14. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, low a similar principle. The additional ligand interactions mediated G. Lipford, H. Wagner, and S. Bauer. 2004. Species-specific recognition of sin- by the TLR9 LRR insertions might induce steric forces that require gle-stranded RNA via Toll-like receptor 7 and 8. Science 303: 1526–1529. more flexibility, and this could be achieved by the low-consensus 15. Ewald, S. E., B. L. Lee, L. Lau, K. E. Wickliffe, G. P. Shi, H. A. Chapman, and G. M. Barton. 2008. The ectodomain of Toll-like receptor 9 is cleaved to generate region between LRR14 and 15 whose structure is unknown, finally a functional receptor. Nature 456: 658–662. resulting in a decrease of the ECD diameter upon CpG-specific 16. Park, B., M. M. Brinkmann, E. Spooner, C. C. Lee, Y. M. Kim, and H. L. Ploegh. binding to TLR9 (11). In our model (Fig. 6A), the very C-terminal 2008. Proteolytic cleavage in an endolysosomal compartment is required for ac- tivation of Toll-like receptor 9. Nat. Immunol. 9: 1407–1414. part of the ECD of TLR9 would be responsible for protein-protein 17. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, Downloaded from interactions resulting in the preformed dimer. This would explain C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. why none of the mutants analyzed (all are located between Immunol. 5: 190–198. LRR-NT and LRR20) influenced dimer formation. This is in con- 18. Kubarenko, A., M. Frank, and A. N. Weber. 2007. Structure-function relation- cordance with the m-shaped dimers observed in the crystal struc- ships of Toll-like receptor domains through homology modelling and molecular dynamics. Biochem. Soc. Trans. 35: 1515–1518. tures of other TLR ligand-receptor complexes, which show indi- 19. Bell, J. K., I. Botos, P. R. Hall, J. Askins, J. Shiloach, D. M. Segal, and vidual ECD converging in the middle with their C termini (5, 9). D. R. Davies. 2005. The molecular structure of the Toll-like receptor 3 ligand- http://www.jimmunol.org/ The conformational change induced in each ECD by engagement binding domain. Proc. Natl. Acad. Sci. USA 102: 10976–10980. 20. Sali, A., and J. P. Overington. 1994. Derivation of rules for comparative protein of the ligand at N- and C-terminal parts of the ECD could be modeling from a database of protein structure alignments. Protein Sci. 3: relayed to the Toll/IL-1 receptor domains via this fixed point of 1582–1596. 21. van der Spoel, D., E. Lindahl, B. Hess, G. Groenhof, A. E. Mark, and contact, leading to a new relative orientation of Toll/IL-1 receptor H. J. Berendsen. 2005. GROMACS: fast, flexible, and free. J. Comput. Chem. 26: domains that enables cytoplasmic signaling. The requirement for 1701–1718. an exact positioning of N- and C-terminal ligand binding patches, 22. Melo, F., D. Devos, E. Depiereux, and E. Feytmans. 1997. ANOLEA: a www server to assess protein structures. Proc. Int. Conf. Intell. Syst. Mol. Biol. 5: LRR insertions and receptor-receptor dimerization regions may 187–190. explain why TLR9 is so sensitive to subtle changes in the LRR 23. Guex, N., and M. C. Peitsch. 1997. SWISS-MODEL and the Swiss-PdbViewer: horseshoe compared with other TLR. Further structural and func- an environment for comparative protein modeling. Electrophoresis 18: by guest on September 29, 2021 2714–2723. tional studies experiments outside the scope of this present work 24. Dolinsky, T. J., J. E. Nielsen, J. A. McCammon, and N. A. Baker. 2004. will be required to unravel the precise determinants of ligand rec- PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electro- statics calculations. Nucleic Acids Res. 32: W665–W667. ognition by TLR9 and other nucleic acid detecting receptors. 25. Li, H., A. D. Robertson, and J. H. Jensen. 2005. Very fast empirical prediction and rationalization of protein pKa values. Proteins 61: 704–721. 26. Baker, N. A., D. Sept, S. Joseph, M. J. Holst, and J. A. McCammon. 2001. Acknowledgments Electrostatics of nanosystems: application to microtubules and the ribosome. We thank J. George for helpful discussions and M. Frank and W. von der Proc. Natl. Acad. Sci. USA 98: 10037–10041. Lieth for computer and modeling assistance. We acknowledge technical 27. Omueti, K. O., J. M. Beyer, C. M. Johnson, E. A. Lyle, and R. I. Tapping. 2005. support from C. Schu¨tz and A. Dillmann. Domain exchange between human Toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination. J. Biol. Chem. 280: 36616–36625. 28. Brown, D. A., S. H. Kang, S. M. Gryaznov, L. DeDionisio, O. Heidenreich, Disclosures S. Sullivan, X. Xu, and M. I. Nerenberg. 1994. Effect of phosphorothioate mod- ification of oligodeoxynucleotides on specific protein binding. J. Biol. Chem. 269: The authors have no financial conflict of interest. 26801–26805. 29. Schotte, P., R. Schauvliege, S. Janssens, and R. Beyaert. 2001. The cathepsin B inhibitor z-FA.fmk inhibits production in macrophages stimulated by References lipopolysaccharide. J. Biol. Chem. 276: 21153–21157. 1. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate 30. Barton, G. M., J. C. Kagan, and R. Medzhitov. 2005. Intracellular localization of immunity. Cell 124: 783–801. Toll-like receptor 9 prevents recognition of self DNA but facilitates access to 2. Gay, N. J., and M. Gangloff. 2007. Structure and function of Toll receptors and viral DNA. Nat. Immunol. 7: 49–56. their ligands. Annu. Rev. Biochem. 76: 141–165. 31. Meng, G., A. Grabiec, M. Vallon, B. Ebe, S. Hampel, W. Bessler, H. Wagner, and 3. Bell, J. K., G. E. Mullen, C. A. Leifer, A. Mazzoni, D. R. Davies, and C. J. Kirschning. 2003. Cellular recognition of tri-/di-palmitoylated peptides is D. M. Segal. 2003. Leucine-rich repeats and pathogen recognition in Toll-like independent from a domain encompassing the N-terminal seven leucine-rich re- receptors. Trends Immunol. 24: 528–533. peat (LRR)/LRR-like motifs of TLR2. J. Biol. Chem. 278: 39822–39829. 4. Jin, M. S., and J. O. Lee. 2008. Structures of the Toll-like receptor family and its 32. Takada, E., S. Okahira, M. Sasai, K. Funami, T. Seya, and M. Matsumoto. 2007. ligand complexes. Immunity 29: 182–191. C-terminal LRRs of human Toll-like receptor 3 control receptor dimerization and 5. Jin, M. S., S. E. Kim, J. Y. Heo, M. E. Lee, H. M. Kim, S. G. Paik, H. Lee, and signal transmission. Mol. Immunol. 44: 3633–3640. J. O. Lee. 2007. Crystal structure of the TLR1-TLR2 heterodimer induced by 33. Weber, A. N. R., M. C. Moncrieffe, M. Gangloff, J. L. Imler, and N. J. Gay. 2005. binding of a Tri-acylated lipopeptide. Cell 130: 1071–1082. Ligand-receptor and receptor-receptor interactions act in concert to activate sig- 6. Kim, H. M., B. S. Park, J. I. Kim, S. E. Kim, J. Lee, S. C. Oh, P. Enkhbayar, naling in the Drosophila Toll pathway. J. Biol. Chem. 280: 22793–22799. N. Matsushima, H. Lee, O. J. Yoo, and J. O. Lee. 2007. Crystal structure of the 34. Hu, X., Y. Yagi, T. Tanji, S. Zhou, and Y. T. Ip. 2004. Multimerization and TLR4-MD-2 complex with bound endotoxin antagonist eritoran. Cell 130: interaction of Toll and Spatzle in Drosophila. Proc. Natl. Acad. Sci. USA 101: 906–917. 9369–9374. 7. Choe, J., M. S. Kelker, and I. A. Wilson. 2005. Crystal structure of human 35. Hendrich, B., and A. Bird. 1998. Identification and characterization of a family Toll-like receptor 3 (TLR3) ectodomain. Science 309: 581–585. of mammalian methyl-CpG binding proteins. Mol. Cell. Biol. 18: 6538–6547. 8. Pirher, N., K. Ivicak, J. Pohar, M. Bencina, and R. Jerala. 2008. A second binding 36. Rutz, M., J. Metzger, T. Gellert, P. Luppa, G. B. Lipford, H. Wagner, and site for double-stranded RNA in TLR3 and consequences for activa- S. Bauer. 2004. Toll-like receptor 9 binds single-stranded CpG-DNA in a se- tion. Nat. Struct. Mol. Biol. 15: 761–763. quence- and pH-dependent manner. Eur. J. Immunol. 34: 2541–2450.