Latrunculin alters the actin-monomer subunit interface to prevent polymerization
Latrunculin alters the actin-monomer subunit interface to prevent polymerization
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DOI：
10.1038/35014075
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发表时间：
2000-06-01
影响因子：
21.3
通讯作者：
McLaughlin, PJ
中科院分区：
文献类型：
--
作者：
Morton, WM;Ayscough, KR;McLaughlin, PJ
†英国格拉斯哥G12 8 QQ，格拉斯哥大学戴维森大楼，生物医学与生命科学研究所电子邮件：Paul。McLaughlin@ ed. ac。uk atrunculin-A是一种能够快速、可逆和特异性破坏肌动蛋白细胞因子1的药物。其作用的功效使其成为许多细胞生物学实验室的首选化合物，取代了经典的肌动蛋白解聚药物细胞松弛素D。其中一个原因是latrunculin的作用模式似乎没有细胞松弛素复杂。而后者影响肌动蛋白丝聚合的动力学在两个倒钩和尖端，latrunculin-A似乎只与肌动蛋白单体，从而防止它们重新聚合成adjecents 2。latrunculin与单体，而不是丝状，肌动蛋白的协会给了我们机会，进一步了解这种相互作用的肌动蛋白单体的详细结构分析，使用晶体学技术。在这里，我们展示了第一个高分辨率的结构与肌动蛋白的肌动蛋白破坏药物，并讨论如何与肌动蛋白的相互作用，以及其结合引起的构象变化，可以解释其在细胞内的作用模式。Latrunculin（图1a）是从Latrunculia magnifians中纯化出来的，Latrunculia magnifians是一种红海海绵，散发出有毒的红色液体，可以在几分钟内杀死鱼类。两个相关的化合物，latrunculin-A和latrunculin-B，从流体中分离，显示出在体外和体内的去甲肌动蛋白结构1，4。体外研究表明，latrunculin仅与肌动蛋白单体结合，并且这种相互作用的动力学与复合物不能聚合一致2。与细胞松弛素不同，latrunculin可以破坏酵母细胞中的肌动蛋白细胞骨架。这使得遗传学研究得以进行，从而促进了肌动蛋白基因中导致细胞对药物作用产生抗性的点突变的鉴定5（图1b）。发现引起latrunculin抗性的突变聚集在一个不同的位点周围，靠近核苷酸结合位点，这表明它们可能鉴定出latrunculin的潜在结合位点。然而，由于该位点并不靠近肌动蛋白丝上的亚基，也不靠近其他与肌动蛋白结合的蛋白质的结合位点，因此，latrunculin发挥其作用的机制尚不清楚。在这类结合蛋白的三个已知例子中，profilin是不合适的，因为它促进核苷酸交换，而脱氧核糖核酸酶1结合到在酵母遗传学研究中与latrunculin结合有关的结构域。与此相反，凝溶胶蛋白结构域1与肌动蛋白6复合，使这些结构域游离，也减少了核苷酸交换，如latrunculin。因此，我们将latrunculin-A
† Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK‡ e-mail: Paul. McLaughlin@ ed. ac. uk atrunculin-A is a drug that is capable of rapidly, reversibly and specifically disrupting the actin cytoskeleton1. The efficacy of its action has made it a compound of choice in many cell-biology laboratories, supplanting the classic actin-depolymerizing drug cytochalasin-D. One reason for this is that the mode of action of latrunculin seems to be less complex than that of cytochalasin. Whereas the latter affects the kinetics of actin-filament polymerization at both the barbed and pointed ends, latrunculin-A seems to associate only with actin monomers, thereby preventing them from repolymerizing into filaments2. The association of latrunculin with monomeric, rather than filamentous, actin gave us the opportunity to further our understanding of this interaction by detailed structural analysis of actin monomers using crystallographic techniques. Here we show the first high-resolution structure of an actin-disrupting drug in association with actin and discuss how its interactions with actin, and the conformational changes that its binding causes, may explain its mode of action within the cell. Latrunculin (Fig. 1a) is purified from Latrunculia magnificans, a Red Sea sponge that exudes a noxious, red fluid that kills fish within minutes3. Two related compounds, latrunculin-A and latrunculin-B, isolated from the fluid were shown to depolymerize actin structures both in vitro and in vivo1, 4. The in vitro studies showed that latrunculin binds only to the actin monomer and that the kinetics of this interaction are consistent with the complex being unable to polymerize2. Unlike cytochalasin, latrunculin can disrupt the actin cytoskeleton in yeast cells. This has enabled genetic studies to be carried out that have facilitated the identification of point mutations in the actin gene that cause cells to become resistant to the effects of the drug5 (Fig. 1b). The mutations that give rise to latrunculin resistance were found to be clustered around a distinct site, close to the nucleotide-binding site, which indicated that they might identify a potential binding site for latrunculin. However, as this site is not close to recognized subunit contacts in the filament, or to known binding sites for other proteins that associate with actin, the mechanism by which latrunculin exerts its effects has remained unclear.Actin has never been known to crystallize in the absence of a binding protein that keeps it in a monodispersed state. Of the three known examples of such binding proteins, profilin is inappropriate as it promotes nucleotide exchange, whereas deoxyribonuclease1 binds to domains that have been implicated, in studies of yeast genetics, in latrunculin binding. In contrast, gelsolin domain 1 in complex with actin6 leaves these domains free and also reduces nucleotide exchange, as does latrunculin. We therefore soaked latrunculin-A