title: Leucine-rich repeat 11 of Toll-like receptor 9 can tightly bind to CpG-containing oligodeoxynucleotides, and the positively charged residues are critical for the high affinity.
abstract: TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-κB activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.
authors: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, 400038 Chongqing, China.
authors: Xichun
authors: X
authors: Pan
authors: Junjie
authors: J
authors: Yue
authors: Guofu
authors: G
authors: Ding
authors: Bin
authors: B
authors: Li
authors: Xin
authors: X
authors: Liu
authors: Xinchuan
authors: X
authors: Zheng
authors: Mengchen
authors: M
authors: Yu
authors: Jun
authors: J
authors: Li
authors: Weiwei
authors: W
authors: Jiang
authors: Chong
authors: C
authors: Wu
authors: Jiang
authors: J
authors: Zheng
authors: Hong
authors: H
authors: Zhou
mesh_terms: D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000276:immunology / Q000494:pharmacology*; D011485:Protein Binding; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; C495371:TLR9 protein, human; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9