Ca-pump and Flippase; transmitting structural changes from catalytic site to transport site

2023 Fiscal Year Final Research Report

• Project/Area Number: 20K06534
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43030:Functional biochemistry-related
• Research Institution: Asahikawa Medical College
• Principal Investigator: Daiho Takashi 旭川医科大学, 医学部, 准教授 (90207267)
• Project Period (FY): 2020-04-01 – 2024-03-31
• Keywords: Caポンプ / リン酸化中間体 / クライオ電子顕微鏡 / エネルギー共役 / 一分子蛍光観察 / ポリアミン / パーキンソン病 / 脂質

Outline of Final Research Achievements

In the EP isomerization / Ca transport of the Ca pump, the A domain on the cytoplasmic side rotates greatly and changes its arrangement, and the structural changes are transmitted to the transport site far away and Ca is released. The specific site of the A domain was fluorescently labeled and observed by a single molecule fluorescence, and its movement was live detected during the EP degradation process, indicating that it passes through a state equivalent to E2PCa2. The Arg324 side chain of the Ca pump M4 changes the binding partner according to the lipid head and reaction stage. This binding has shown multifunctionality that plays an important role in EP isomerization.
We elucidated the atomic structure of the intermediate by cryoelectron microscopy of the enzyme ATP13A2 similar to flippase, and clarified the transport mechanism from the structural changes in polyamine binding and transport associated with E2P hydrolysis and Pi release, as well as site-specific mutations.

Free Research Field

生化学

Academic Significance and Societal Importance of the Research Achievements

CaポンプのEP異性化/Ca輸送；Ca2E1P → Ca2E2P → E2Pでは、Aドメインが駆動する細胞質領域の配置変化が伝達されCa放出する。Ca2E2Pの存在は未実証であった。Aドメインを蛍光ラベルし、その動きの観察から野生型CaポンプのEP異性化でCa2E2P状態をライブ検知した。CaポンプのArg324側鎖が反応段階に応じて脂質頭部と結合し、EP異性化に重要な役割を持つことを示した。ポリアミンを輸送するATP13A2はパーキンソン病の原因遺伝子PARK9である。輸送サイクルの各中間体のクライオ電顕構造を解明し、輸送機構を明らかにした。上記疾患の遺伝的な原因と治療法の探求に貢献する。