D-loop of Actin Differently Regulates the Motor Function of Myosins II and V
D-loop of Actin Differently Regulates the Motor Function of Myosins II and V
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DOI：
10.1074/jbc.m109.013565
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发表时间：
2009-12-11
影响因子：
4.8
通讯作者：
Ishiwata, Shin'ichi
中科院分区：
文献类型：
--
作者：
Kubota, Hiroaki;Mikhailenko, Sergey V.;Ishiwata, Shin'ichi
为了获得更多关于肌动蛋白-肌球蛋白相互作用方式的信息，我们研究了肌球蛋白II和V的运动特性如何受到肌动蛋白的DNase I结合环(D-loop)修饰的影响，这两种修饰以两种不同的方式进行，即枯草杆菌蛋白水解酶和M47A点突变。在体外动力分析中，这两种修饰都显著降低了肌球蛋白II-重肌球蛋白上的滑动速度，这是由于产生的力较弱，但增加了它在肌球蛋白V上的滑动速度。另一方面，肌球蛋白V的单分子在野生型和修饰肌动蛋白上以相同的速度“行走”；但是，行程长度急剧缩短，这与D-环修饰导致肌球蛋白对肌动蛋白的亲和力降低有关。肌球蛋白V的单分子测量和整体测量之间的差异表明，在体外运动分析中，非配位的多个肌球蛋白V分子通过与相同的肌动蛋白细丝结合而相互施加内耗，而内耗因与修饰的肌动蛋白结合较弱而减少。这些结果表明，D-环强烈调节肌球蛋白II的作用力和肌球蛋白V的加工能力，可能影响两种肌球蛋白的肌动蛋白-ADP中心点P-I状态下的肌动蛋白-肌球蛋白相互作用。
To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V "walked" with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP center dot P-i state of both myosins.