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

## TL;DR

This study explores how the Msp1 enzyme uses energy to extract proteins from cell membranes, revealing insights into its mechanism and energy requirements.

## Contribution

The study introduces covalently linked dimers to dissect the energetic and mechanistic roles of Msp1 subunits in substrate extraction.

## Key findings

- Msp1 dimers assembled into active pseudohexameric trimers, showing E193Q does not block ATP hydrolysis.
- Substrate extraction was impaired in some constructs, indicating position-specific effects of subunit activity.
- High ATPase rates showed minimal impact on substrate extraction, suggesting excess ATPase capacity.

## Abstract

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.

Cells must clear mislocalized or faulty proteins from membranes to survive. The AAA+ ATPase Msp1 performs this task, but dissecting how its six subunits work together is challenging. We engineered linked dimers with varied numbers of functional subunits to reveal how Msp1 subunits cooperate and use energy to extract proteins from the lipid bilayer.