Load-dependent destabilization of the γ-rotor shaft in F _O F ₁ ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

Abstract

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

Catalytic robustness and torque generation of the F1-ATPase

“…On the other hand, some studies suggest that the stalks are twisted in the ATP synthase at work. Although the elasticity of the peripheral stalk has been reported previously (Junge et al 2009), recent studies suggest that the γ subunit of the central stalk is twisted (Okazaki and Hummer 2015; Vahidi et al 2016). It remains elusive how the elasticity of the stalks contribute to energy transduction within the ATP synthase.…”

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

“…The group of Lars Konermann did pioneering work on the Fo‐F1 ATP synthase from E. coli . They observed the changes in structural dynamics of the molecular motor in cation in native inverted vesicles directly extracted from E. coli (Table ).…”

Mass spectrometry-enabled structural biology of membrane proteins

“…Interestingly, GPCRs solubilized in bicelles yielded better sequence coverage in HDX-MS workflows than those solubilized in detergent [199]. The conformational dynamics of the F 0 F 1 ATP synthase during catalysis have also been studied by HDX-MS using protein solubilized in inside-out membrane vesicles, revealing that the rotor shaft is destabilized when pumping protons against a transmembrane gradient [194]. More recently, the SMALP platform was implemented to study the rhomboid protease GlpG in different lipid conditions, to identify regions of the protein sensitive to the lipid environment [195].…”