Motions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model
Motions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model
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DOI：
10.1016/s0022-2836(03)00178-5
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发表时间：
2003-03-28
影响因子：
5.6
通讯作者：
Freemont, PS
中科院分区：
文献类型：
--
作者：
Beuron, F;Flynn, TC;Freemont, PS
p97是属于AAA（与各种细胞活性相关的ATP酶）蛋白超家族的Mg-ATP酶，已经提出在两种不同的细胞途径中起作用，即通过利用不同的接头复合物进行同型膜融合和泛素蛋白降解。我们目前的冷冻电子显微镜三维重建内源性p97在AMP-PNP结合状态在24 A的分辨率。它揭示了明确的核苷酸依赖性的差异相比，我们以前发表的“p97-ADP”重建，包括一个惊人的重排的N结构域和两个ATP酶结构域，D1和D2，相对于彼此的位置变化。ADP结合状态下N-D1结构域的X射线结构的对接表明，N结构域的向上重新定位是必要的，以适应“p97-AMP-PNP”的冷冻-EM图，这表明在核苷酸水解时N结构域的取向发生了变化。此外，计算分析的变形运动的p97，进行的cryo-EM密度图和原子结构的N-D1域独立，显示存在的负协同性之间的D1和D2环和灵活性的N域。总之，这些结果允许识别功能上重要的功能，提供分子的见解到拟议的p97分子伴侣功能的动态。(C)2003爱思唯尔科技有限公司版权所有。
p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic membrane fusion and ubiquitin protein degradation by utilizing differing adaptor complexes. We present the cryo-electron microscopy three-dimensional reconstruction of endogenous p97 in an AMP-PNP bound state at 24 A resolution. It reveals clear nucleotide-dependent differences when compared to our previously published "p97-ADP" reconstruction, including a striking rearrangement of N domains and a positional change of the two ATPase domains, D1 and D2, with respect to each other. The docking of the X-ray structure of N-D1 domains in an ADP bound state indicates that an upward repositioning of N domain is necessary to accommodate the cryo-EM map of "p97-AMP-PNP", suggesting a change in the orientation of N domains upon nucleotide hydrolysis. Furthermore, computational analysis of the deformational motions of p97, performed on the cryo-EM density map and the atomic structure of the N-D1 domains independently, shows the existence of a negative cooperativity between the D1 and D2 rings and the flexibility of the N domains. Together these results allow the identification of functionally important features that offer molecular insights into the dynamics of the proposed p97 chaperone function. (C) 2003 Elsevier Science Ltd. All rights reserved.