Centre for Structural Biology, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.
Open Biol. 2014 Mar 5;4(3):130142. doi: 10.1098/rsob.130142.
The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.
II 型 AAA+ 蛋白 p97 参与众多细胞活动，包括内质网相关降解、转录激活、膜融合和细胞周期调控。这些活动至少部分受到泛素系统的调节，其中 p97 被认为是在大分子复合物内靶向泛素化蛋白底物，并协助其提取或解体。尽管 ATP 酶活性对 p97 的功能至关重要，但对于 ATP 结合或水解如何与 p97 构象变化和底物重塑相偶联，人们知之甚少。在这里，我们使用单颗粒电子 cryoEM（冷冻电镜）研究核苷酸对 p97 构象的影响。我们已经从 cryo-EM 数据集的识别出构象异质性，从中我们解析了两种主要的 p97 构象。对构象的比较揭示了核苷酸结合和水解时的环间旋转，这可能与靶蛋白复合物的重塑有关。
