Molecular and Cellular Biophysics Program, University of North Carolina, Chapel Hill, NC 27599.
Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Mol Biol Cell. 2018 Feb 1;29(3):285-294. doi: 10.1091/mbc.E17-01-0057. Epub 2017 Nov 29.
XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain's architecture and localization mechanism remain outstanding. We present the crystal structure of the Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer's N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.
XMAP215/Dis1 家族蛋白是强有力的微管聚合酶，对于有丝分裂纺锤体的结构和动态至关重要。虽然微管聚合酶的活性是由 N 端肿瘤过度表达基因（TOG）结构域阵列驱动的，但适当的细胞定位是充分发挥活性的必要条件，这是由 C 端结构域介导的。C 端结构域的结构和定位机制的结构洞察力仍然存在。我们展示了 Stu2 C 端结构域的晶体结构，揭示了一个 15nm 的平行同源二聚体卷曲螺旋。卷曲螺旋的平行结构对微管聚合过程中同源二聚体 N 端 TOG 结构域的排列具有机械意义。该卷曲螺旋有两个空间上不同的保守区域：CRI 和 CRII。CRI 和 CRII 中的突变会扰乱 Stu2 在有丝分裂纺锤体中的分布和定位，并导致纺锤体形态缺陷，包括错位和碎片化纺锤体的频率增加。总的来说，这些数据突出了 Stu2 二聚化结构域作为因子结合支架的作用，它可以将 Stu2 最佳地定位在有丝分裂纺锤体上，以促进适当的纺锤体结构和动态。
