Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
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DOI：
10.1038/s41598-020-58070-y
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发表时间：
2020-01-23
影响因子：
4.6
通讯作者：
Iino, Ryota
中科院分区：
文献类型：
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作者：
Ando, Jun;Shima, Tomohiro;Iino, Ryota
细胞质动力蛋白是一种沿沿着运动的二聚体动力蛋白。其马达结构域（头部）水解ATP并诱导接头、茎和微管结合结构域（MTBD）的构象变化以触发步进运动。在这里，我们应用金纳米粒子（金纳米粒子）的散射成像可视化无负荷步进运动的进行性动力蛋白。我们观察到人工二聚嵌合动力蛋白，其具有来自盘基网柄藻细胞质动力蛋白的头部、接头和柄，以及来自人轴丝动力蛋白的MTBD，其结构已通过冷冻电子显微镜进行了充分研究。一个头部的二聚体标记有30纳米金纳米粒子，步进运动观察到100 μ s的时间分辨率和亚纳米定位精度在生理相关的1 mM ATP。我们发现8 nm的向前和向后的步骤和5 nm的侧步骤，与微管上α β-微管蛋白二聚体之间的结合裂缝的轴上和轴外间距一致。前步的概率是后步的1.8倍，与侧步的概率相近。没有清楚地观察到单头束缚态，并且步骤受到单个速率常数的限制。我们的研究结果表明，动力蛋白主要移动有偏见的小步进运动，其中只有向后的步骤略有抑制。
Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 mu s time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between alpha beta-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed.