The Boulder Laboratory for 3-D Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.
PLoS One. 2013;8(1):e53792. doi: 10.1371/journal.pone.0053792. Epub 2013 Jan 14.
We have used cryo-electron microscopy (cryo-EM) and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1's nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor's coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.
我们使用冷冻电子显微镜（cryo-EM）和螺旋平均法来研究异二聚体 kinesin-14 Kar3Vik1 与微管结合的 3D 结构，分辨率为 2.5nm。在 Kar3Vik1 的核苷酸水解循环的关键点获得了 3D 图谱，以深入了解该马达用于逆行运动的机制。在 Kar3Vik1 与微管保持强烈相互作用的所有状态下，我们发现，正如冷冻电镜观察到的那样，马达与一个头部结构域结合，而第二个头部向外延伸。Kar3Vik1-微管复合物的 3D 重建显示，在无核苷酸状态下，马达的卷曲螺旋茎指向微管的正极。在 ATP 状态下，外头被观察到经历了一个大的旋转，将茎旋转约 75°，指向微管的负极。为了确定在每个核苷酸状态下哪一个头部与微管蛋白结合，我们采用了 Vik1 的特异性 Nanogold®标记。得到的图谱证实，在无核苷酸、ATP 和 ADP+Pi 状态下，Kar3 与微管表面保持接触，而 Vik1 远离微管并随着卷曲螺旋的旋转追踪，当它向微管负极旋转时。虽然许多先前的研究都集中在同源二聚体 kinesin 的机制上，但这项工作首次全面研究了异二聚体 kinesin 的动力冲程。这里显示的茎旋转高度类似于报道的同源二聚体 kinesin-14 Ncd 的旋转，强调了负向运动机制的保守性。
