Hoenger A, Sack S, Thormählen M, Marx A, Müller J, Gross H, Mandelkow E
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
J Cell Biol. 1998 Apr 20;141(2):419-30. doi: 10.1083/jcb.141.2.419.
We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results with the x-ray crystal structure of monomeric and dimeric kinesin. A monomeric kinesin construct (rK354, containing only a short neck helix insufficient for coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin head per tubulin subunit (alpha-beta-heterodimer). The orientation of the kinesin head (an anterograde motor) on the microtubule surface is similar to that of ncd (a retrograde motor). A longer kinesin construct (rK379) forms a dimer because of the longer neck helix forming a coiled-coil. Unexpectedly, this construct also decorates the microtubule with a stoichiometry of one head per tubulin subunit, and the orientation is similar to that of the monomeric construct. This means that the interaction with microtubules causes the two heads of a kinesin dimer to separate sufficiently so that they can bind to two different tubulin subunits. This result is in contrast to recent models and can be explained by assuming that the tubulin-kinesin interaction is antagonistic to the coiled-coil interaction within a kinesin dimer.
我们用单体和二聚体驱动蛋白构建体修饰微管，通过冷冻电子显微镜和三维图像重建研究它们的结构，并将结果与单体和二聚体驱动蛋白的X射线晶体结构进行比较。一种单体驱动蛋白构建体（rK354，仅包含一个不足以形成卷曲螺旋的短颈部螺旋）以每微管蛋白亚基（α-β异二聚体）一个驱动蛋白头部的化学计量比修饰微管。驱动蛋白头部（一种正向运动蛋白）在微管表面的方向与ncd（一种反向运动蛋白）相似。由于较长的颈部螺旋形成卷曲螺旋，一个更长的驱动蛋白构建体（rK379）形成二聚体。出乎意料的是，该构建体也以每微管蛋白亚基一个头部的化学计量比修饰微管，并且方向与单体构建体相似。这意味着与微管的相互作用使驱动蛋白二聚体的两个头部充分分离，从而它们可以结合到两个不同的微管蛋白亚基上。这一结果与最近的模型相反，并且可以通过假设微管蛋白-驱动蛋白相互作用与驱动蛋白二聚体内的卷曲螺旋相互作用相拮抗来解释。
