p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured ‘power-stroke’-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.

p97 处理泛素化底物并在细胞蛋白质稳态中发挥核心作用。在这里，我们报告了一系列与底物结合的人类 p97 复合物的低温电子显微镜结构，分辨率范围为 2.9 到 3.8 Å，捕获了 p97 的 D1 和 D2 ATPase 环的“动力冲程”样运动。这些结构的一个关键特征是亚基间信号 (ISS) 基序的关键构象变化，它加强了核苷酸和邻近亚基的结合，并有助于 D1 和 D2 环的螺旋楼梯构象。此外，我们以 2.4 Å 的分辨率确定了人 p97 与 NMS-873（一种有效的 p97 抑制剂）复合物的冷冻电镜结构。结构显示 NMS-873 结合在 D2 结构域的一个隐蔽凹槽处并与 ISS 基序相互作用，