Energetic requirements and mechanistic plasticity in Msp1-mediated substrate extraction from lipid bilayers

AAA+ proteins are essential molecular motors involved in numerous cellular processes, yet their mechanism of action in extracting membrane proteins from lipid bilayers remains poorly understood. One roadblock for mechanistic studies is the inability to generate subunit specific mutations within these hexameric proteins. Using the mitochondrial AAA+ protein Msp1 as a model, we created covalently linked dimers with varying combinations of wild type and catalytically inactive E193Q mutations. The wide range of ATPase rates in these constructs allows us to probe how Msp1 uses the energy from ATP hydrolysis to perform the thermodynamically unfavorable task of removing a transmembrane helix (TMH) from a lipid bilayer. Our in vitro and in vivo assays reveal a non-linear relationship between ATP hydrolysis and membrane protein extraction, suggesting a minimum ATP hydrolysis rate is required for effective TMH extraction. While structural data often supports a sequential clockwise/2-residue step (SC/2R) mechanism for ATP hydrolysis, our biochemical evidence suggests mechanistic plasticity in how Msp1 coordinates ATP hydrolysis between subunits, potentially allowing for robustness in processing challenging substrates. This study enhances our understanding of how Msp1 coordinates ATP hydrolysis to drive mechanical work and provides foundational insights about the minimum energetic requirements for TMH extraction and the coordination of ATP hydrolysis in AAA+ proteins.