Structural characterization of a dynein motor domain.
Structural characterization of a dynein motor domain.
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动力蛋白运动域的结构表征。
DOI：
10.1006/jmbi.1997.1584
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Koonce,MP
中科院分区：
文献类型：
--
作者：
Samso,M;Radermacher,M;Frank,J;Koonce,MP
细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起重要作用。作为一种分子马达，动力蛋白利用ATP水解机制结合和释放微管，并发生构象变化，导致微管负端的净位移。为了可视化这种动力蛋白的结构特征，我们已经开始用电子显微镜和图像处理来表征动力蛋白头部结构域。用透射电镜对Dictyostelium负染色的天然动力蛋白进行了观察，并用软件SPIDER对头部区域的图像进行了对齐和分析。由此产生的二维平均值显示出一个长圆形，由七到八个球体域或裂片组成，环绕着一个充满污渍的区域。动力蛋白重链的380 kDa重组片段仅编码球状头结构域；对这些粒子的分析表明，它们在结构上与原头部结构域具有很高的相似性。在该片段的几个突出部分可以看到一个突出的茎，表明其结构类似于某些轴突动力蛋白的b链。单倾斜对图像用于计算低分辨率三维重建的动力头域。这显示了一个长度为13.5 nm的扁平球体形状，七个相似的区域排列在一个环中。重建的切片显示一个大的中央腔。这是对动力蛋白分子头部结构的首次详细描述。中心空腔的存在和外部的球形特征，以及它的大尺寸使得动力蛋白在结构上与肌凝蛋白或运动蛋白不同。
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.