Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway

• Published in: Nature communications Vol. 15; no. 1; pp. 7505 - 11
• Main Authors: Cooney, Ian, Schubert, Heidi L., Cedeno, Karina, Fisher, Olivia N., Carson, Richard, Price, John C., Hill, Christopher P., Shen, Peter S.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 29.08.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 101/28; 631/45/173; 631/45/612/1981; 631/45/612/645; 631/535/1258/1259; 82/58; Adenosine triphosphatase; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; ATP; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - metabolism; Cellular structure; Enzymes; Humanities and Social Sciences; Hydrolysis; Intracellular Signaling Peptides and Proteins; Models, Molecular; Motor task performance; multidisciplinary; Protein Binding; Protein folding; Protein transport; Protein Unfolding; Proteins; Quality control; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Science; Science (multidisciplinary); SHP-1 protein; Substrates; Translocation; Valosin Containing Protein - chemistry; Valosin Containing Protein - genetics; Valosin Containing Protein - metabolism; Yeasts
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-51835-3

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a ‘hand-over-hand’ mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding. The Cdc48 enzyme is an abundant and essential enzyme that functions in many cellular pathways as a protein unfoldase. Here, the authors determine an ensemble of Cdc48 structures that capture snapshots of its unfolding action using a ‘hand-over-hand’ mechanism.