Samsó M, Radermacher M, Frank J, Koonce M P
Department of Biomedical Sciences, State University of New York, Albany 12201-0509, USA.
J Mol Biol. 1998 Mar 13;276(5):927-37. doi: 10.1006/jmbi.1997.1584.
Cytoplasmic dynein is a microtubule-based mechanochemical protein that plays an essential role in cell division, vesicle transport, and cytoplasmic membrane organization. As a molecular motor, dynein utilizes an ATP hydrolysis mechanism to bind and release microtubules and to undergo conformational changes that result in a net displacement towards the microtubule's minus end. To visualize structural features of this motor protein, we have begun to characterize the dynein head domain by electron microscopy and image processing. Transmission electron microscopy of negatively stained native dynein from Dictyostelium has been performed and images of the head domain have been aligned and analyzed with the software SPIDER. The resulting 2D averages show an oblong round shape composed of seven to eight globular domains or lobes that encircle a stain-filled area. A recombinant 380 kDa fragment of the dynein heavy chain encodes just the globular head domain; analysis of these particles reveals a high structural similarity with the native head domain. A prominent stalk can be seen in several projections of this fragment, suggesting a structure analogous to the B-link described for some axonemal dyneins. Single tilt pair images were used to compute low resolution 3D reconstructions of the dynein head domain. These show a flattened spheroidal shape of 13.5 nm in length with seven similar domains arranged in a ring. Slices through the reconstructions reveal a large central cavity. This is the first detailed description of the head domain structure for a dynein molecule. The presence of a central cavity and the outer globular features, along with its large size make dynein structurally distinct from either myosin or kinesin.
胞质动力蛋白是一种基于微管的机械化学蛋白，在细胞分裂、囊泡运输和细胞质膜组织中发挥着至关重要的作用。作为一种分子马达，动力蛋白利用ATP水解机制来结合和释放微管，并经历构象变化，从而导致向微管负端的净位移。为了观察这种马达蛋白的结构特征，我们已开始通过电子显微镜和图像处理来表征动力蛋白头部结构域。已对盘基网柄菌的负染天然动力蛋白进行了透射电子显微镜观察，并使用SPIDER软件对头部结构域的图像进行了对齐和分析。所得的二维平均值显示出一个椭圆形，由七到八个球状结构域或叶组成，围绕着一个充满染色剂的区域。动力蛋白重链的一个重组380 kDa片段仅编码球状头部结构域；对这些颗粒的分析揭示了与天然头部结构域高度的结构相似性。在该片段的几个投影中可以看到一个突出的柄，表明其结构类似于一些轴丝动力蛋白所描述的B链。单倾斜对图像用于计算动力蛋白头部结构域的低分辨率三维重建。这些重建显示出一个扁平的球形，长度为13.5 nm，有七个相似的结构域排列成一个环。通过重建的切片显示出一个大的中央腔。这是对动力蛋白分子头部结构域结构的首次详细描述。中央腔和外部球状特征的存在，以及其较大的尺寸，使得动力蛋白在结构上与肌球蛋白或驱动蛋白截然不同。
