Structural studies of nucleic acid sensing Toll-like receptors

• Project/Area Number: 25291010
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Shimizu Toshiyuki 東京大学, 薬学研究科(研究院), 教授 (30273858)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥18,070,000 (Direct Cost: ¥13,900,000、Indirect Cost: ¥4,170,000); Fiscal Year 2015: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2014: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2013: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
• Keywords: Toll様受容体 / 自然免疫 / X線結晶解析 / 一本鎖核酸 / １本鎖核酸 / X線結晶構造解析 / Ｘ線結晶構造解析

Outline of Final Research Achievements

Innate immunity serves as the first line of defense against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses.
TLR8 recognizes single stranded (ss) RNA derived from bacterial and viral DNA. We solved the crystal structure of TLR8 in complex with ssRNA and revealed that TLR8 is a uridine senso. TLR8 can be synergistically activated by uridine and oligonucleotide degraded from ssRNATLR9 recognizes bacterial and viral DNA containing the Cytosine-phosphate-Guanine (CpG) dideoxynucleotide motif. Here we show the crystal structure of TLR9 bound to agonistic CpG-DNA, forming a symmetric TLR9/CpG-DNA complex with 2:2 stoichiometry. CpG-DNA was recognized by both protomers in the dimer, in particular by the N-terminal fragment (LRRNT-LRR10) from one protomer and the C-terminal fragment (LRR20-LRR22) from the other.

Research Products

• [Int'l Joint Research] University of Minnesota(米国)
• [Journal Article] Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9. (2015)
  • Author(s): Ohto, U., Shibata, T., Tanji, H., Krayukhina, E., Uchiyama, S., Miyake, K., and Shimizu, T.
  • Journal Title: Nature Volume: 520 Issue: 7549 Pages: 702-705
  • DOI: 10.1038/nature14138
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] DNase II-dependent DNA digestion is required for DNA sensing by TLR9 (2015)
  • Author(s): Chan MP, Onji M, Fukui R, Kawane K, Shibata T, Saitoh SI, Ohto U, Shimizu T, Barber GN, *Miyake K
  • Journal Title: Nat Commun Volume: 6 Issue: 1 Pages: 5853-5853
  • DOI: 10.1038/ncomms6853
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Targeting cell surface TLR7 for therapeutic intervention in autoimmune diseases (2015)
  • Author(s): Kanno A, Tanimura N, Ishizaki M, Ohko K, Motoi Y, Onji M, Fukui R, Shimozato T, Yamamoto K, Shibata T, Sano S, Sugahara-Tobinai A, Takai T, Ohto U, Shimizu T, Saitoh S, Miyake K.
  • Journal Title: Nature Communications Volume: 6 Issue: 1 Pages: 6119-6119
  • DOI: 10.1038/ncomms7119
  • Peer Reviewed / Open Access
• [Journal Article] Structure-based Design of Human TLR8-specific Agonists with Augmented Potency and Adjuvanticity. (2015)
  • Author(s): Beesu, M., Caruso, G., Salyer, A., Khetani, K., Sil, D., Weerasinghe, M., Tanji, H., Ohto, U., Shimizu, T., & David, S. A.
  • Journal Title: J. Med. Chem. Volume: 58 Issue: 19 Pages: 7833-7849
  • DOI: 10.1021/acs.jmedchem.5b01087
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Toll-like receptor 8 senses degradation products of single-stranded RNA. (2015)
  • Author(s): Tanji H, Ohto U, Shibata T, Taoka M, Yamauchi Y, Isobe T, Miyake K,
  • Journal Title: Nat Struct Mol Biol. Volume: 22 Issue: 2 Pages: 109-115
  • DOI: 10.1038/nsmb.2943
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Determinants of activity at human Toll-like receptors 7 and 8: quantitative structure-activity (2014)
  • Author(s): Yoo E, Salunke DB, Sil D, Guo X, Salyer AC, Hermanson AR, Kumar M,
  • Journal Title: J Med Chem. Volume: 57 Issue: 19 Pages: 7955-7970
  • DOI: 10.1021/jm500744f
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Structure and function of toll-like receptor 8. (2014)
  • Author(s): Ohto, U., Tanji, H., & Shimizu, T.
  • Journal Title: Microbes Infect. Volume: 16 Issue: 4 Pages: 273-282
  • DOI: 10.1016/j.micinf.2014.01.007
  • Peer Reviewed
• [Journal Article] Structural reorganization of the Toll-like receptor 8 dimer induced by agonistic ligands (2013)
  • Author(s): Tanji H, Ohto U, Shibata T, Miyake K, Shimizu T
  • Journal Title: Science Volume: 339 Issue: 6126 Pages: 1426-1429
  • DOI: 10.1126/science.1229159
  • Peer Reviewed
• [Journal Article] Human TLR4 polymorphism D299G/T399I alters TLR4/MD-2 conformation and response to a weak ligand monophosphoryl lipid A (2013)
  • Author(s): N. Yamakawa, U. Ohto, S. Akashi-Takamura, K. Takahashi, SI. Saitoh, N. Tanimura, T. Suganami, Y. Ogawa, T. Shibata, T. Shimizu, K. Miyake
  • Journal Title: Int. Immunol. Volume: 25 Issue: 1 Pages: 45-52
  • DOI: 10.1093/intimm/dxs084
  • Peer Reviewed
• [Presentation] Structural studies of single-stranded RNA receptor TLR8 (2015)
  • Author(s): Hiromi Tanji, Umeharu Ohto, Takuma Shibata, Kensuke Miyake, Toshiyuki Shimizu
  • Organizer: Toll meeting 2015
  • Place of Presentation: Palacio de Congresos de Mabella(SPAIN)
  • Year and Date: 2015-10-02
  • Int'l Joint Research
• [Presentation] 自然免疫系一本鎖RNA受容体TLR8の構造基盤 (2015)
  • Author(s): 丹治裕美、大戸梅治、柴田琢磨、田岡万悟、山内芳雄、礒邉俊明、三宅健介、清水敏之
  • Organizer: 第15回日本蛋白質科学会年会
  • Place of Presentation: あわぎんホール（徳島県徳島市）
  • Year and Date: 2015-06-25
• [Presentation] Structural study of TLR8 sensing single stranded RNA in innate immune system (2014)
  • Author(s): Shimizu T
  • Organizer: The Cold Spring Harbor Asia conference
  • Place of Presentation: Suzhou Dushu Lake Conference Center, Suzhou, China
  • Year and Date: 2014-10-27 – 2014-10-31
  • Invited
• [Presentation] Structural study of TLR8 sensing single stranded RNA in innate immune system (2014)
  • Author(s): Shimizu T
  • Organizer: The 4th Asia Pacific Protein Association (APPA) Conference
  • Place of Presentation: Jeju, Korea
  • Year and Date: 2014-05-17 – 2014-05-20
• [Presentation] Toll 様受容体 8(TLR8)の構造科学的研究 (2013)
  • Author(s): 清水敏之
  • Organizer: 日本生化学大会
  • Place of Presentation: パシフィコ横浜（神奈川県）
• [Presentation] Structural study of innate immune receptor, TLR
  • Author(s): Shimizu T
  • Organizer: FAOBMB Mini-Symposium 2013
  • Place of Presentation: 岩手医科大学（岩手県）
  • Invited
• [Remarks] 蛋白構造生物学教室
  • URL: http://www.f.u-tokyo.ac.jp/~kouzou/