Tropomyosin-binding site(s) on the Dictyostelium actin surface as identified by site-directed mutagenesis.
Tropomyosin-binding site(s) on the Dictyostelium actin surface as identified by site-directed mutagenesis.
复制标题
通过定点诱变鉴定出盘基网柄菌肌动蛋白表面上的原肌球蛋白结合位点。
作者：
K. Saeki;K. Sutoh;T. Wakabayashi
为了鉴定肌动蛋白分子表面上的原肌球蛋白结合位点，我们检查了肌动蛋白子结构域 4 引入诱变的效果。由于盘基网柄菌肌动蛋白的Gln228-Ser232序列与不结合原肌球蛋白的四膜虫肌动蛋白的Gln228-Ser232序列不同，因此产生了盘基网柄菌/四膜虫嵌合肌动蛋白。此外，Lys238 和 Glu241 被替换为丙氨酸（突变体 645），以研究位于 β-折叠两端的带电残基的作用。作为对照实验，在 N 末端附近引入负电荷（突变体 663）。为了便于分离突变肌动蛋白而不影响正常功能，将Glu360替换为组氨酸。作为此类突变体的对照突变体，产生了突变体647(E360H)。突变肌动蛋白在盘基网柄菌属细胞中表达。所有突变肌动蛋白均具有功能：它们 (i) 聚合并 (ii) 激活兔骨骼肌球蛋白亚片段 1 (S1) 的 ATP 酶活性。突变体 663 (G2E) 显示出原肌球蛋白结合并激活肌球蛋白 ATP 酶，几乎与兔骨骼肌动蛋白一样。然而，突变体 645 (K238A/E241A/E360H) 的原肌球蛋白结合变得镁依赖性。嵌合肌动蛋白（突变体 646：QTAAS-KAYKE 替换和 E360H）即使在镁离子存在下也显示出原肌球蛋白结合减少。这些结果表明“开启”状态肌动蛋白的原肌球蛋白结合位点位于子结构域4上。令人惊讶的是，在原肌球蛋白-肌钙蛋白存在的情况下，嵌合肌动蛋白比兔骨骼肌动蛋白显示出更多的协同钙调节。在原肌球蛋白-肌钙蛋白存在的情况下，无论钙浓度如何，突变体肌动蛋白 645 都很难激活 S1 ATP 酶，尽管该肌动蛋白本身可以激活 S1 ATP 酶。讨论了细丝调节的空间阻断或协同/变构机制。
To identify tropomyosin-binding site(s) on the surface of actin molecule, we examined the effect of mutagenesis introduced to subdomain 4 of actin. Because the sequence of Gln228-Ser232 of Dictyostelium actin differs from that of Tetrahymena actin that does not bind tropomyosin, the Dictyostelium/Tetrahymena chimeric actin was produced. Also, Lys238 and Glu241 were replaced with alanine (mutant 645) to study the role of charged residues which are located at both ends of a beta-sheet. As a control experiment, a negative charge was introduced near to the N-terminus (mutant 663). To facilitate the separation of mutant actins without affecting the normal function, Glu360 was replaced with histidine. As a control mutant to such mutants, the mutant 647 (E360H) was produced. Mutant actins were expressed in Dictyostelium cells. All mutant actins were functional: they (i) polymerize and (ii) activate ATPase activity of rabbit skeletal myosin subfragment-1 (S1). The mutant 663 (G2E) showed tropomyosin binding and activated myosin ATPase almost as well as rabbit skeletal actin. However, the tropomyosin binding of the mutant 645 (K238A/E241A/E360H) became magnesium dependent. The chimeric actin (mutant 646: QTAAS-to-KAYKE replacement and E360H) showed decreased tropomyosin binding even in the presence of magnesium ions. These results indicate that the tropomyosin-binding sites of "on"-state actin are on subdomain 4. Surprisingly, the chimeric actin showed more cooperative calcium regulation than rabbit skeletal actin in the presence of tropomyosin-troponin. The mutant actin 645 can hardly activate S1 ATPase irrespective of calcium concentration in the presence of tropomyosin-troponin, even though this actin by itself can activate S1 ATPase. The steric blocking or cooperative/allosteric mechanism of thin filament regulation is discussed.