Nogales E, Wolf S G, Khan I A, Ludueña R F, Downing K H
Life Science Division, Lawrence Berkeley Laboratory, California 94720, USA.
Nature. 1995 Jun 1;375(6530):424-7. doi: 10.1038/375424a0.
Tubulin, the major component of microtubules, is a heterodimer of two chains, alpha and beta, both of relative molecular mass 50,000 (Mr50K) and with 40-50% identity. The isotypic variety and conformational flexibility of tubulin have so far made it impossible to obtain crystals for X-ray work. Structural knowledge of tubulin has been limited to about 20 A from X-ray diffraction of oriented microtubules, and from electron microscopy of microtubules and zinc-induced crystalline sheets in negative stain. The sheets consist of protofilaments similar to those in microtubules but associated in an antiparallel arrangement, and their two-dimensional character is ideal for high-resolution electron microscopy. Here we present a three-dimensional reconstruction of tubulin to 6.5 A resolution, obtained by electron crystallography of zinc-induced two-dimensional crystals of the protein. The alpha- and beta-subunits appear topologically similar, in agreement with their sequence homology. Several features can be defined in terms of secondary structure. An apparent alpha-helical portion, adjacent to both interdimer and inter-protofilament contacts, is tentatively attributed to a segment near the carboxy terminus of the protein. We can assign the alpha- and beta-subunits on the basis of projection studies of the binding of taxol, which show one taxol site per tubulin heterodimer, in agreement with the known stoichiometry of taxol in microtubules. These studies indicate that taxol affects the interaction between protofilaments; to our knowledge, this is the first time that a ligand-binding site has been visualized in the tubulin molecule.
微管蛋白是微管的主要成分，是由α和β两条链组成的异二聚体，二者相对分子质量均为50,000（Mr50K），序列一致性为40 - 50%。迄今为止，微管蛋白的同型多样性和构象灵活性使得无法获得用于X射线研究的晶体。微管蛋白的结构知识一直局限于通过定向微管的X射线衍射、微管的电子显微镜观察以及负染条件下锌诱导的晶体片层所获得的约20埃的信息。这些片层由类似于微管中原纤维的结构组成，但呈反平行排列，其二维特性非常适合高分辨率电子显微镜观察。在此，我们通过对该蛋白锌诱导的二维晶体进行电子晶体学分析，展示了分辨率达6.5埃的微管蛋白三维重构。α和β亚基在拓扑结构上相似，这与它们的序列同源性相符。可以根据二级结构定义几个特征。一个明显的α螺旋部分，毗邻二聚体间和原纤维间的接触点，初步认为归因于该蛋白羧基末端附近的一个片段。我们可以根据紫杉醇结合的投影研究来确定α和β亚基，该研究表明每个微管蛋白异二聚体有一个紫杉醇结合位点，这与微管中已知的紫杉醇化学计量相符。这些研究表明紫杉醇影响原纤维之间的相互作用；据我们所知，这是首次在微管蛋白分子中观察到配体结合位点。
