Discovery of the fastest myosin, its amino acid sequence, and structural features

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 ( Cc XI) 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 ( Cb XI-1, 2, 3, and 4) and measured their velocities. While the velocities of Cb XI-3 and Cb XI-4 were similar to that of Cc XI, the velocities of Cb XI-1 and Cb XI-2 were estimated to be 73 and 66 μm s−1, respectively, suggesting that Cb XI-1 and Cb XI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that Cb XI-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. ### Competing Interest Statement The authors have declared no competing interest.