Characterizing the full length Npl4-Ufd1 complex and interaction with Cdc48 through an interface residue switch

A core element of the protein quality control (PQC) and protein degradation process is Cdc48 (VCP/p97), a highly conserved AAA-ATPase, along with its canonic cofactors, Ufd1 and Npl4 (UN). Here, we introduce novel structural insights into the interactions of the Cdc48-Ufd1-Npl4 ternary complex. We combined high resolution cross-linking mass spectrometry (XL-MS) with integrative modeling to map the interaction between Ufd1 and Npl4, in the absence and presence of Cdc48. We show that the dynamics of UN assembly is embedded in Cdc48 binding. Characterization of the Cdc48-UN complex pointed to a set of important binding residues, one of which, cysteine 115, is in the multiple binding domains of Cdc48 with diverse UBX cofactors. Mutation of this cysteine to serine alters the stability of the ternary complex, which resulted in decreased interactions of the Cdc48-UN complex in vitro, along with a collapse in cellular growth and protein quality control in yeast. This work defined Cdc48’s mode of interaction and can be used for development of therapeutics mediating protein quality control.