Evidence for a Partially Stalled γ Rotor in F₁-ATPase from Hydrogen-Deuterium Exchange Experiments and Molecular...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• F1-ATPase utilizes ATP hydrolysis to power the rotation of its gamma (γ) subunit.
• The γ subunit's C-terminal helix acts as the rotor tip within the apical bearing formed by α3β3.
• The precise conformation of the γ subunit during rotation and its deviation from crystal structures are not fully understood.

Purpose of the Study:

• To investigate the conformational dynamics of the γ subunit during F1-ATPase rotation.
• To explore the mechanical forces and potential unfolding events of the γ subunit's C-terminal helix.
• To compare experimental findings with existing models of F1-ATPase rotation.

Main Methods:

• Hydrogen-deuterium exchange (HDX) mass spectrometry was employed to study E. coli F1-ATPase.
• Molecular dynamics (MD) simulations were developed to model off-axis forces on the γ subunit.
• HDX kinetics and MD-predicted hydrogen bond opening events were correlated.

Main Results:

• Rotation of the γ subunit led to increased deuteration in its C-terminal helix, indicating conformational flexibility.
• HDX data suggests the rotor tip occasionally unfolds, rather than rotating smoothly as in traditional models.
• MD simulations revealed rotor tip stalling and unfolding, consistent with experimental HDX patterns.

Conclusions:

• F1-ATPase operation involves significant rotational resistance, causing sporadic unfolding of the γ C-terminal helix.
• The rotor tip's fragility, unlike the smooth rotation predicted by the "greasy bearing" model, is linked to the absence of the c10 ring.
• The combined MD/HDX strategy offers a powerful approach for studying molecular motor mechanisms.