The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.

Automated summary

Cdc48 (VCP/p97) is a key AAA-ATPase involved in protein quality control, together with Ufd1 and Npl4 (UN). This study reveals the novel structural insights into the interactions within the Cdc48-Npl4Ufd1 complex. By using integrative modeling and crosslinking mass spectrometry, the interaction between Npl4 and Ufd1, alone and in complex with Cdc48, is mapped. The study provides structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex and its implications in vivo.