Discovery of the fastest myosin, its amino acid sequence, and structural features
Discovery of the fastest myosin, its amino acid sequence, and structural features
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DOI：
10.1101/2021.05.06.442907
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发表时间：
2021-05
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影响因子：
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通讯作者：
Takeshi Haraguchi;M. Tamanaha;Kano Suzuki;K. Yoshimura;Takuma Imi;M. Tominaga;H. Sakayama;T. Nishiyama;T. Murata;Kohji Ito
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作者：
Takeshi Haraguchi;M. Tamanaha;Kano Suzuki;K. Yoshimura;Takuma Imi;M. Tominaga;H. Sakayama;T. Nishiyama;T. Murata;Kohji Ito
在轮藻（Chara）的细胞中，细胞质以极高的速度（~70 μm s−1）流动。由于细胞质流动是由细胞器相关的肌球蛋白XI沿着沿着肌动蛋白丝滑动引起的，因此有人认为轮藻细胞中存在肌球蛋白XI，其速度为70 μm s-1，是迄今为止测量到的最快的肌球蛋白。然而，先前克隆的轮藻肌球蛋白XI（CcXI）以大约20 μm s-1的速度移动肌动蛋白丝，这表明轮藻中可能存在一种未知的肌球蛋白XI，其速度为70 μm s-1。最近，布朗轮藻的基因组序列已经公布，揭示了这种轮藻有四个肌球蛋白XI基因。在本文报道的工作中，我们克隆了这四个肌球蛋白XI（CbXI-1，2，3和4），并测量了它们的速度。虽然CbXI-3和CbXI-4的速度与CcXI相似，但CbXI-1和CbXI-2的速度估计分别为73和66 μm s-1，这表明GbXI-1和GbXI-2是轮藻细胞胞质流动的主要贡献者，并表明GbXI-1是迄今为止发现的最快的肌球蛋白。我们还报告了第一个原子结构（2.8 μ m分辨率）的肌球蛋白XI使用X射线晶体学。基于这种晶体结构和最近发表的低分辨率（4.3 μ m）的肌动蛋白-肌球蛋白XI的cryo-EM结构，似乎肌动蛋白结合区域有助于轮藻肌球蛋白XI的快速运动。肌动蛋白结合表面环2的突变实验支持这一假设。50多年来，人们一直认为生物界中最快的肌球蛋白（速度为70 μm s-1）存在于轮藻中，因为轮藻细胞中的细胞质流动速度为70 μm s-1。然而，具有这种速度的肌球蛋白尚未被鉴定。在这项工作中，我们成功地克隆了一个速度为73 μm s-1的肌球蛋白XI，这是迄今为止测量到的最快的肌球蛋白。我们还首次成功地结晶了肌球蛋白XI。结构分析和突变实验表明，定义轮藻肌球蛋白XI的快速运动的中心区域是肌动蛋白结合位点。
Cytoplasmic streaming with extremely high velocity (~70 μm s−1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 μm s−1, the fastest myosin measured so far, exists in Chara cells. However, the previously cloned Chara corallina myosin XI (CcXI) moved actin filaments at a velocity of around 20 μm s−1, suggesting that an unknown myosin XI with a velocity of 70 μm s−1 may be present in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 were similar to that of CcXI, the velocities of CbXI-1 and CbXI-2 were estimated to be 73 and 66 μm s−1, respectively, suggesting that CbXI-1 and CbXI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that CbXI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis. Significance statement It has been suggested for more than 50 years that the fastest myosin in the biological world, with a velocity of 70 μm s−1, exists in the alga Chara because cytoplasmic streaming with a velocity of 70 μm s−1 occurs in Chara cells. However, a myosin with that velocity has not yet been identified. In this work, we succeeded in cloning a myosin XI with a velocity of 73 μm s−1, the fastest myosin so far measured. We also successfully crystallized myosin XI for the first time. Structural analyses and mutation experiments suggest that the central regions that define the fast movement of Chara myosin XI are the actin-binding sites.