Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin
Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin
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DOI：
10.1073/pnas.0910787106
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发表时间：
2009-12-22
影响因子：
11.1
通讯作者：
Yamamoto, Keiichi
中科院分区：
文献类型：
--
作者：
Ito, Kohji;Yamaguchi, Yukie;Yamamoto, Keiichi
大多数肌球蛋白有一个带正电荷的环2，环2上有一簇赖氨酸残基，与带负电荷的肌动蛋白N端片段结合。然而，非常快的轮藻肌球蛋白的环2的净电荷为零，其中没有赖氨酸簇。相反，轮藻肌球蛋白有一个高正电荷的环3。为了阐明轮藻肌球蛋白在其高速和高肌动蛋白激活的ATP酶活性的这些独特的表面环的作用，我们进行了突变分析，使用重组轮藻肌球蛋白马达结构域。结果表明，第3环的净正电荷影响肌动蛋白激活ATP酶的V-max和K-app，而对ATP酶的速度影响不大。环2中的净正电荷影响K-app和速度，但不影响V-max。我们的研究结果表明，轮藻肌球蛋白已经进化到具有高正电荷的环3，其高ATP酶活性和具有较少的正电荷的环2，其高速度。由于环3中的高正电荷和环2中的低正电荷似乎是轮藻肌球蛋白的高速度的原因之一，我们操纵网囊藻肌球蛋白（II类）的环2和3中的电荷含量。去除网囊藻肌球蛋白环2的正电荷并在环3中加入正电荷使其运动速度高于野生型，表明环2和环3中的电荷策略具有广泛的适用性。
Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected V-max and K-app of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K-app and the velocity, although it did not affect V-max. Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.