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 homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. 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 the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex. The human AAA+protein p97 plays an important role in cellular protein homeostasis. Here, the authors use cryo-EM to obtain further insights into how p97 interacts with its co-factor Npl4 and they observe three distinct conformational states of Npl4 in complex with human p97, which suggests that a seesaw motion is essential for the unfolding activity of the p97 complex.

Automated summary

The human AAA+protein p97 plays a crucial role in cellular protein homeostasis. The study reveals insights into the interaction between p97 and its co-factor Npl4, showing three distinct conformational states of Npl4 in complex with human p97, suggesting the essential role of seesaw motion in the unfolding activity of the p97 complex.