Meurer-Grob P, Kasparian J, Wade R H
Institut de Biologie Structurale (CEA/CNRS), 41 rue Jules Horowitz, 38027 Grenoble, France.
Biochemistry. 2001 Jul 10;40(27):8000-8. doi: 10.1021/bi010343p.
Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.
微管结构会因真核生物种类、细胞类型以及稳定剂的存在而有所不同。对于体外组装的微管， sarcodictyin A、埃坡霉素B和刺参素（与紫杉醇类似）的存在会减少原纤维的平均数量，但多西他赛会增加原纤维的平均数量。已知用缓慢水解的GTP类似物GMPCPP组装可得到96%的14原纤维微管。我们使用电子冷冻显微镜和螺旋重建技术，获得了多西他赛和GMPCPP微管的三维图谱，数据分辨率达到14 Å。通过将微管蛋白晶体结构对接至这些改进的微管图谱中，来检查微管壁内的二聚体堆积情况。对接的微管蛋白和从“原子分辨率”微管模型计算出的模拟图像显示，微管蛋白异源二聚体沿原纤维首尾相连排列，β亚基位于微管正端。两种类型的微管中，相邻原纤维内微管蛋白二聚体的相对位置相同，这证实了微管蛋白亚基之间保守的横向相互作用是不同微管结构表面晶格适应性的原因。体内存在的具有非常规原纤维数量的微管，其表面晶格组织可能与体外发现的相同。类微管解聚蛋白诱导的弯曲“GDP”微管蛋白构象似乎会削弱微管蛋白亚基之间的横向接触，并可能阻止微管组装或促进微管解聚。我们得出结论，相邻原纤维中微管蛋白亚基之间的横向接触对微管的稳定性、刚性和结构起决定性作用。
