Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis1. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development4. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that under oxidative conditions, the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) inhibits p97 function by releasing cupric ions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.