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Updated: Jun 14, 2025
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Purification of Hsp104, a Protein Disaggregase
Published on: September 30, 2011
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Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway
Ian Cooney1, Heidi L Schubert1, Karina Cedeno1
1Department of Biochemistry, University of Utah, Salt Lake City, UT, USA.
Nature Communications
|August 29, 2024
Summary
The Cdc48 enzyme, crucial for protein quality control, uses a hand-over-hand mechanism for substrate unfolding. Its D1 and D2 domains coordinate ATP hydrolysis and substrate engagement in a sequential cycle.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Cdc48 AAA+ ATPase is vital for protein quality control, featuring two motor domains (D1 and D2) in a hexameric ring structure.
- Understanding the dynamic mechanism of Cdc48 in substrate unfolding is critical for elucidating its role in cellular proteostasis.
Purpose of the Study:
- To determine the structural basis of Cdc48-mediated substrate unfolding in complex with the adaptor Shp1.
- To elucidate the mechanism of substrate translocation and unfolding by visualizing the enzyme throughout its functional cycle.
Main Methods:
- Affinity purification of native Cdc48-Shp1 complexes from budding yeast lysate.
- Ensemble structural analysis using cryo-electron microscopy (or similar structural biology techniques) to capture various states of the translocation cycle.
Main Results:
- A continuum of structural snapshots revealing the entire substrate translocation cycle was obtained.
- Detailed interactions between Shp1 and Cdc48 were uncovered, supporting a hand-over-hand mechanism.
- Sequential ATP hydrolysis and substrate engagement by D1 and D2 domains were observed, with D2 playing a dominant role.
Conclusions:
- The study reveals a coordinated, sequential hand-over-hand mechanism for Cdc48-mediated substrate unfolding.
- The distinct roles of D1 and D2 motors in ATP hydrolysis and substrate interaction explain the enzyme's function in protein quality control.
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