Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA.
J Struct Biol. 2013 Nov;184(2):335-44. doi: 10.1016/j.jsb.2013.09.020. Epub 2013 Oct 4.
Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.
Kar3Cik1 是酿酒酵母中的一种异二聚体驱动蛋白-14，参与有丝分裂中的纺锤体形成和交配细胞中的核融合。Kar3 代表一个典型的驱动蛋白马达结构域，在 ATP 水解的控制下与微管相互作用。在体内，Kar3 的定位和功能受到与其相互作用的 Cik1 或 Vik1 的调节，Cik1 和 Vik1 是两个密切相关的运动同源结构域，缺乏核苷酸结合位点。事实上，Vik1 在结构上类似于驱动蛋白头部的核心。尽管密切相关，但 Kar3Cik1 和 Kar3Vik1 各自负责体内的一组独特功能，并且在体外也表现出不同的生化行为。为了确定它们不同功能能力的结构基础，我们使用冷冻电子显微镜和螺旋重建技术研究了各种核苷酸状态下与微管结合的 Kar3Cik1 复合物的 3-D 结构，并将我们的 3-D 数据与 Kar3Vik1 和同源二聚体驱动蛋白-14 Ncd 的数据进行了比较。由于缺乏 Cik1 运动同源结构域的 X 射线晶体结构，我们基于与相关的 Vik1、Kar3 和 Ncd 的序列相似性预测了该 Cik1 结构域的结构。通过分子对接到我们的 3-D 图谱中，我们产生了一个详细的近原子模型，显示了处于两种不同核苷酸状态下的 Kar3Cik1 与微管的复合物，一种是无核苷酸状态，另一种是 ATP 结合状态。我们的数据表明，尽管它们的功能不同，但异二聚体 Kar3Cik1 和 Kar3Vik1 以及同源二聚体 Ncd 在不同的核苷酸状态下都具有惊人的结构相似性，这表明在驱动蛋白-14 家族中存在共同的机制主题。
