Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
J Biol Chem. 2010 Aug 13;285(33):25591-601. doi: 10.1074/jbc.M110.123869. Epub 2010 Jun 8.
The nucleotide state of actin (ATP, ADP-P(i), or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165-172) changes from an unstructured loop to a beta-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277-1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P(i) state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.
肌动蛋白的核苷酸状态（ATP、ADP-P(i)或 ADP）已知会影响其在聚合时与其他肌动蛋白分子的相互作用，以及在肌动蛋白单体和丝状状态下与多种肌动蛋白结合蛋白的相互作用。最近，分子动力学模拟预测，位于亚结构域 1 和 3 界面的一段序列（W 环；残基 165-172）在 ATP 水解时从无规卷曲环转变为β-转角构象（Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277-1283）。该区域直接参与 F-肌动蛋白与其他亚基的结合，以及与丝切蛋白、原肌球蛋白和 WH2 结构域蛋白的结合，因此可能有助于这些相互作用的核苷酸敏感性。本研究证明了 W 环区域与肌动蛋白核苷酸结合裂隙之间的相互交流。残基 167、169 和 170 的点突变及其定点标记显著影响核苷酸从裂隙区域的释放，而 ATP/ADP 转换改变位于 W 环的探针的荧光。在 ADP-P(i)状态下，W 环采用与 ATP 状态相似但与 ADP 状态不同的构象。藤黄菌素 A 与核苷酸裂隙的结合有利于 W 环的 ATP 样构象，而 Arg-177 的 ADP-核糖基化迫使 W 环形成与 ADP 和 ATP 状态不同的构象。总体而言，我们的实验数据表明，肌动蛋白的 W 环是一个核苷酸传感器，可能有助于 F-肌动蛋白的核苷酸状态依赖性变化，以及 G-和 F-肌动蛋白与肌动蛋白结合蛋白的核苷酸状态调节相互作用。
