substrate-induced product-release mechanism of L-PGDS

• Project/Area Number: 16K18868
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Physical pharmacy
• Research Institution: Kindai University
• Principal Investigator: SHIMAMOTO SHIGERU 近畿大学, 理工学部, 講師 (00610487)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2018: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: プロスタグランジンD2 / 等温滴定型熱測定 / 酵素活性測定 / 構造解析 / PGDS / 核磁気共鳴法 / プロスタグランジンD合成酵素 / プロスタグランジンD / リポカリン / 睡眠 / 輸送体型酵素

Outline of Final Research Achievements

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic ligands in the extracellular space, and is known as the PGD2-synthesizing enzyme responsible for the sleep regulation. The binding affinity and stoichiometry to each ligand were analyzed by isothermal titration calorimetry (ITC), and the binding region for each ligand on L-PGDS was estimated by NMR titration experiment. The ITC results showed that PGD2 and PGF2α bound to L-PGDS with a stoichiometry of 2 to 1 but PGE2 with a stoichiometry of 1 to 1. In addition, the NMR experiments indicated that PGD2 and PGF2α bound to both the catalytic site containing the Cys65 and non-catalytic site of L-PGDS, while PGE2 bound to only the non-catalytic site.

Academic Significance and Societal Importance of the Research Achievements

L-PGDSの酵素反応モデルで、基質・生成物が2分子結合することは全く考慮されてこなかった。本研究では、1）従来のモデルにない新しい概念（2つの結合サイト・生成物放出）を加えた新規酵素反応モデルが構築し、これまで着目されていなかった親和性の低い結合サイトでの基質認識が生成物の産生速度に影響を与えることを示した。2）L-PGDSの活性中心のみをターゲットにした阻害剤の効果が弱い原因を解明し、さらに、新規結合部位における基質や生成物側の認識に重要な部位の同定とタンパク質側の認識に関与するアミノ酸を同定した。これらの情報は、新たに生成物放出メカニズムをターゲットとした新規薬剤の開発につながるだろう。

Research Products

• [Journal Article] Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4 (2019)
  • Author(s): Yamamoto Kenji、Ishibashi Osamu、Sugiura Keisuke、Ubatani Miki、Sakaguchi Masaya、Nakatsuji Masatoshi、Shimamoto Shigeru、Noda Masanori、Uchiyama Susumu、Fukutomi Yuma、Nishimura Shigenori、Inui Takashi
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 1503-1503
  • DOI: 10.1038/s41598-018-38134-w
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] ITC Analysis of Ligand Binding to Lipocalin-type Prostaglandin D Synthase. (2017)
  • Author(s): Shimamoto S., Maruno T.
  • Journal Title: Netsu Sokutei Volume: 44 Pages: 108-116
  • NAID: 40021281379
  • Peer Reviewed
• [Presentation] Molecular recognition mechanism of Hematopoietic Prostaglandin D Synthase with cofactor and its substrate (2018)
  • Author(s): Shimamoto Shigeru
  • Organizer: 62nd Annual Meeting Biophysical Society
  • Int'l Joint Research
• [Presentation] Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for its product. (2017)
  • Author(s): Shigeru Shimamoto, Yusuke Nakagawa, Yuta Nakahata,Takahiro Maruno, Yuji Kobayashi, Kosuke Aritake, Yoshihiro Urade, and Yuji Hidaka
  • Organizer: 61st Annual Meeting Biophysical Society
  • Place of Presentation: New Orleans, Louisiana
  • Year and Date: 2017-02-11
  • Int'l Joint Research