Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates
Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates
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DOI：
10.1021/bi972903j
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发表时间：
1998-05-12
期刊：
影响因子：
2.9
通讯作者：
Spudich, JA
中科院分区：
文献类型：
--
作者：
Murphy, CT;Spudich, JA
虽然肌球蛋白运动结构域的大部分序列在各种生物体和组织类型中高度保守，但 25 和 50 kDa 结构域以及 50 和 20 kDa 结构域之间的连接却截然不同。 50-20K 环位于与肌动蛋白相互作用的位置，而 25-50K 环位于更靠近 ATP 结合位点的位置 [Rayment, I., et al. (1993) 科学 261, 50-58]。 50-20K 环的嵌合研究 [Uyeda, T. Q.-P., et al. (1994) 自然 368, 567-569;罗夫纳，A.S.，等人。 (1995) J.Biol。化学。 [270 (51), 30260-30263]已经表明该环影响肌动蛋白对 ATP 酶活性的激活。鉴于肌球蛋白作为分子马达的功能，有人提出25-50K环可能特异性地改变ADP释放[Spudich, J. A. (1994) Nature 374. 515-518]。在这里，我们通过工程改造含有盘基网柄菌肌球蛋白重链的嵌合体来研究该环的作用，该嵌合体具有来自两种酶促不同的肌球蛋白（兔骨骼肌球蛋白和棘阿米巴肌球蛋白）的环。嵌合肌球蛋白在体内补充肌球蛋白无效表型，正常结合核苷酸，与肌动蛋白正常相互作用，并表现出野生型水平的肌动蛋白激活的ATP酶活性。然而，肌球蛋白释放 ADP 的速率（通常是参与运动的最慢步骤）的变化反映了供体肌球蛋白的活性。总之，盘基网柄菌肌球蛋白重链嵌合体的研究表明，50-20K 序列特异性影响肌动蛋白激活的 ATP 酶活性 [Uyeda, T. Q.-P., et al. 2017]。 (1994)]而25-50K序列有助于确定ADP释放速率。
While most of the sequence of myosin's motor domain is highly conserved among various organisms and tissue types, the junctions between the 25 and 50 kDa domains and the 50 and 20 kDa domains are strikingly divergent. The 50-20K loop is positioned to interact with actin, while the 25-50K loop is situated nearer the ATP binding site [Rayment, I., et al. (1993) Science 261, 50-58]. Chimeric studies of the 50-20K loop [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-569; Rovner, A. S., et al. (1995) J. Biol. Chem. 270 (51), 30260-30263] have shown that this loop affects actin activation of ATPase activity. Given the function of myosin as a molecular motor, it was proposed that the 25-50K loop might specifically alter ADP release [Spudich, J. A. (1994) Nature 374. 515-518]. Here we study the role of this loop by engineering chimeras containing the Dictyostelium myosin heavy chain with loops from two enzymatically diverse myosins, rabbit skeletal and Acanthamoeba. The chimeric myosins complement the myosin null phenotype in vivo, bind nucleotide normally, interact normally with actin, and display wild-type levels of actin-activated ATPase activity. However, the rate of ADP release from the myosins, normally the slowest step involved in motility, was changed in a manner that reflects the activity of the donor myosin. Ln summary, studies of Dictyostelium myosin heavy chain chimeras have shown that the 50-20K sequence specifically affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994)] while the 25-50K sequence helps determine the rate of ADP release.