Identification of an N-Terminal Recognition Site in TLR9 That Contributes to CpG-DNA-Mediated Receptor 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 References This article cites 36 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/182/12/7690.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 protein, 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 glycoproteins 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.