Sokolova Anna V, Kreplak Laurent, Wedig Tatjana, Mücke Norbert, Svergun Dmitri I, Herrmann Harald, Aebi Ueli, Strelkov Sergei V
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry and Institute of Crystallography, Russian Academy of Sciences, Moscow 119991, Russia.
Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16206-11. doi: 10.1073/pnas.0603629103. Epub 2006 Oct 18.
Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris.HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coefficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A(11) mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.
中间丝（IFs）与微管、微丝及相关的交联蛋白一起，构成了后生动物细胞的细胞骨架。虽然最近已经获得了关于代表基本IF“构建块”的二聚体的晶体学数据，但对于成熟IF结构及其组装途径的结构细节知之甚少。在这里，我们应用溶液小角X射线散射技术，通过系统改变离子强度条件，研究了53 kDa的人IF蛋白波形蛋白在pH 8.4时的体外组装，并通过电子显微镜和分析超速离心对这些实验进行了补充。虽然5 mM Tris.HCl（pH 8.4）中的波形蛋白溶液主要含有四聚体，但加入20 mM NaCl会诱导进一步的横向组装，沉降系数的变化证明了这一点，并产生了一种独特的八聚体中间体。四个八聚体最终结合形成单位长度丝（ULFs），这些丝纵向退火。基于补充了晶体学数据和其他结构限制的小角X射线散射实验，构建了波形蛋白四聚体、八聚体和ULF的三维分子模型。在这三种寡聚体中的每一种中，相邻的二聚体仅以近似半交错的反平行A(11)模式排列，它们的轴之间的距离为3.2 - 3.4 nm。ULF似乎是一种动态的、相对松散堆积的结构，其横截面上的质量分布大致均匀。
