The role of multiple ATP-binding sites in dynein motor domain

动力蛋白运动结构域中多个 ATP 结合位点的作用

• 批准号: 12480196
• 负责人: TOYOSHIMA Yoko
• 金额: $ 8.83万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480196/
• 关键词: dynein; ATP binding site; P-loops; Tetrahyemena; inner arms; monomeric dynein; microtubules; Michaelis-Menten; 細胞質ダイニン; 機能ドメイン; 単頭; ATPase; 繊毛運動

Dynein is a huge complex molecule including heavy chain(s) which have multiple (4) ATP binding consensus sequences (P-loops) as well as intermediate and light chains. We purified a monomeric form of dynein (dynein-a) from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein-a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP. Both ATPase activity and transnational velocities in the presence of ADP fit the Michaelis- Menten equation well. However in the absence of ADP, neither of the activities followed the Michaelis-Menten-type kinetics probably due to the effect of two-ATP binding sites. Our results also indicate that dynein-a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system.

动力蛋白是一种巨大的复杂分子，包括具有多个（4个）ATP结合共有序列（P环）的重链以及中间链和轻链。我们从嗜热四膜虫的14 S动力蛋白中分离纯化了一种动力蛋白单体（dynein-a），并对其进行了表征。ATP酶活性在1 mM ATP的存在下，在低水平的ADP的两倍。ATP酶活性和跨膜速度均符合米氏方程.而在ADP不存在时，两种酶的活性均不符合米氏动力学，这可能是由于两个ATP结合位点的作用。我们的研究结果还表明，动力蛋白-a有一个ATP结合位点，这是非常敏感的ADP和影响ATP水解的催化位点。这项研究表明，单体形式的动力蛋白分子通过直接与ATP和ADP结合来调节其活性，因此动力蛋白分子具有分子内调节系统。

项目成果

Uemura, S., Toyoshima, Y.Y, et al.: "Kinesin-microtubule binding is dependent on both nucleotide state and loading direction"Proc. Natl. Acad. Sci. USA. (in press). (2002)

Uemura, S.、Toyoshima, Y.Y 等人：“驱动蛋白-微管结合取决于核苷酸状态和加载方向”Proc.

Yajima, J., Toyoshima, Y.Y., et al.: "Direct long-term observation of kinesin processivity at no load"Current Biol.. 12. 301-306 (2002)

Yajima, J.、Toyoshima, Y.Y. 等人：“无负载下驱动蛋白持续合成能力的直接长期观察”Current Biol.. 12. 301-306 (2002)

Toyoshima,Y.Y., et al.: "Processive movement of single 22S dynein molecules occurs only at low ATP concentrations."Proc.Natl.Acad.Sci.USA. 97. 2533-2537 (2000)

Toyoshima, Y.Y. 等人：“单个 22S 动力蛋白分子的持续运动仅发生在低 ATP 浓度下。”Proc.Natl.Acad.Sci.USA。

Yajima, J., Toyoshima, Y. Y., et al.: "Direct long-term observation of kinesin processivity at no load"Current Biol.. 12. 301-306 (2002)

Yajima, J.、Toyoshima, Y. Y. 等人：“无负载下驱动蛋白持续合成能力的直接长期观察”Current Biol.. 12. 301-306 (2002)

Uemura, S., Toyoshima, Y.Y., et al.: "Kinesin-microtubule binding is dependent on both nucleotide state and loading direction"Proc.Natl.Acid.Sei.USA. (in press). (2002)

Uemura, S.、Toyoshima, Y.Y. 等人：“驱动蛋白-微管结合取决于核苷酸状态和加载方向”Proc.Natl.Acid.Sei.USA。