Linked dimers of the <scp>AAA</scp> + <scp>ATPase</scp> Msp1 reveal energetic demands and mechanistic plasticity for substrate extraction from lipid bilayers

Msp1 is a membrane-anchored AAA+ (ATPases Associated with diverse cellular Activities) enzyme that extracts membrane proteins from lipid bilayers. To understand how the subunits in the homohexamer convert ATP hydrolysis into mechanical work, we developed covalently linked dimers combining wild-type and catalytically inactive (E193Q) subunits. These assembled into pseudohexameric trimers of dimers and retained ATPase activity, indicating that E193Q does not act as a dominant negative for ATP hydrolysis. Conversely, substrate extraction was impaired in some constructs, suggesting position-specific effects. Surprisingly, constructs with a twofold difference in ATPase rates showed minimal differences in substrate extraction across lipid environments, suggesting excess ATPase capacity. These findings clarify how Msp1 coordinates hydrolysis, its energetic requirements, and substrate access to the pore.