Applying Structural Mass Spectrometry to characterize Redox Dependent Interactome of CDC48

The final stage of protein synthesis is protein folding and localization. Control over a healthy proteome begins with the birth of the polypeptide chain and ends with the coordinated death of the mature protein by degradation. One of the major players in eukaryotic protein degradation is the essential and highly conserved ATPase known as CDC48 in yeast (VCP/p97 in mammals). CDC48 serves as a shuttle bus, extracting and delivering misfolded proteins from membranes of the ER, mitochondria, nucleolus and cytosol to the proteasome for degradation.

Previous research in the lab established a link between CDC48’s activity and the ability of cells to cope with oxidative stress, which leads to protein aggregation in cells. Specifically, we identified a novel thiol-switch site, at a highly conserved cysteine 115, which mediates this redox-dependent activity of CDC48 via its localization in the cell.

To dissect redox-regulation of CDC48 on its working cycle and interactome, we implemented a toolbox of different structural approaches to monitor changes in the CDC48’s structure upon oxidation and alteration of the cysteine 115 site. Using a variety of different structural mass spectrometry-based techniques including HDX-MS and cross-linking (XL-MS) coupled with circular dichroism spectroscopy, we defined the structural plasticity of the CDC48’s N-terminal domain harboring cysteine 115 as well as interaction sites with CDC48’s canonical co-factors, Npl4 and Ufd1. Outcomes of this study provides a deeper understanding of the role of the thiol-switch site on structural plasticity and function of CDC48.