Abstract

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Previous studies have showed that it is a potential drug target for cancer, neurodegenerative disease, and viral infections. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of substrate-engaged human p97 complex 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 inter-subunit signaling (ISS) motifs, which tightens the binding of nucleotides and neighboring subunits, and contributes to the spiral staircase conformation of the D1 and D2 rings. We further determined the cryo-EM structure of human p97 in complex with NMS-873, the most potent p97 inhibitor. 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, thus blocking substrate translocation allosterically.