Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
J Cell Biol. 2012 Jun 25;197(7):957-70. doi: 10.1083/jcb.201201132.
Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.
驱动蛋白-14 分子马达产生微管负端指向力，用于有丝分裂和减数分裂。这些分子马达是二聚体，采用非循环动力冲程机制工作，但第二个头在运动中的作用尚不清楚。在酿酒酵母中，驱动蛋白-14 分子 Kar3 与 Vik1 或 Cik1 形成异二聚体。Vik1 含有一个马达同源结构域，保留了微管结合特性，但缺乏核苷酸结合位点。在这种情况下，两个头都与运动有关。在这里，我们通过结构测定、电子显微镜、平衡结合和运动学分析表明，在周期开始时，Kar3Vik1 结合或封闭相邻原丝上的两个 αβ-微管蛋白亚基。当 Vik1 与微管碰撞时，周期开始，随后 Kar3 与微管结合并释放 ADP，从而破坏 Vik1-微管相互作用并为动力冲程的开始定位马达。结果表明，头对头的通讯是通过相邻的卷曲螺旋介导的。
