Departments of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
J Biol Chem. 2013 Jul 5;288(27):19785-95. doi: 10.1074/jbc.M113.463695. Epub 2013 May 17.
Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.
中心纺锤体是动物细胞胞质分裂的关键调节因子。它由 ZEN-4/MKLP1（一种驱动蛋白-6）和 CYK-4/MgcRacGAP（一种 Rho GTP 酶激活蛋白）组成，形成四聚体。在后期，中心纺锤体定位于平行微管重叠的狭窄区域，并启动中心纺锤体的组装。中心纺锤体的组装需要 ZEN-4 和 CYK-4 之间的复合物形成。然而，CYK-4 与 ZEN-4 结合所导致的结构变化以及微管的捆绑机制尚不清楚。在这里，我们研究了 CYK-4 结合是否会诱导 ZEN-4 的构象变化。通过连续波电子顺磁共振（EPR）和双电子电子共振（DEER）光谱学对 ZEN-4 和 CYK-4 之间最小相互作用区域的结构和构象动力学进行表征，结果表明 CYK-4 结合显著稳定了 ZEN-4 颈环区的相对位置。此外，我们的数据表明，在结合和未结合状态下，每个颈环区的结构相似。CYK-4 结合降低了 ZEN-4 介导的微管滑行的速度。这些结果限制了中心纺锤体分子组织的模型。
