Interaction of the mitotic inhibitor monastrol with human kinesin Eg5
Interaction of the mitotic inhibitor monastrol with human kinesin Eg5
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DOI：
10.1021/bi026716j
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发表时间：
2003-01-21
期刊：
影响因子：
2.9
通讯作者：
Kozielski, F
中科院分区：
文献类型：
--
作者：
DeBonis, S;Simorre, JP;Kozielski, F
来自智人的微管依赖性驱动蛋白样蛋白 Eg5 参与有丝分裂纺锤体的组装。它显示了一个三域结构，包括 N 端运动域、中央卷曲线圈和 C 端尾域。体内 HsEg5 受到 monastrol 的可逆抑制，monastrol 是一种细胞可渗透的小分子，可导致细胞有丝分裂停滞。为了定义 HsEg5 上的最小 monastrol 结合域，对单体和二聚体 Eg5 构建体进行了检查。 NMR 弛豫实验表明 monastrol 与本研究中使用的所有 Eg5 构建体相互作用。酶技术表明 monastrol 通过直接与运动结构域结合来部分抑制 Eg5 ATP 酶活性。这种结合对于微管来说是非竞争性的，表明 monastrol 不会干扰运动-MT 复合物的形成。这种结合与 ATP 不具有竞争性。酶学和体内测定均表明 monastrol 的 S 对映体比 R 对映体和外消旋 monastrol 更具活性。停流荧光测定表明 monastrol 通过形成 Eg5-ADP-monastrol 三元复合物来抑制 ADP 释放。 Monastrol 可逆地抑制人 Eg5 的运动。 Monastrol 对驱动蛋白超家族的以下成员没有抑制作用：MC5（果蝇 Ncd）、HK379（智人常规驱动蛋白）、DKH392（果蝇常规驱动蛋白）、BimCl-428（构巢曲霉 BimC）、Klp15（秀丽隐杆线虫 C 末端）马达）或 Nkin460GST（粗糙脉孢菌常规驱动蛋白）。
The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimCl-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin).