Mechanochemical Energy Transduction during the Main Rotary Step in the Synthesis Cycle of F₁-ATPase

Abstract

F-ATPase is a highly efficient molecular motor that can synthesize ATP driven by a mechanical torque. Its ability to function reversibly in either direction requires tight mechanochemical coupling between the catalytic domain and the rotating central shaft, as well as temporal control of substrate binding and product release. Despite great efforts and significant progress, the molecular details of this synchronized and fine-tuned energy conversion mechanism are not fully understood. Here, we use extensive molecular dynamics simulations to reconcile recent single-molecule experiments with structural data and provide a consistent thermodynamic, kinetic and mechanistic description of the main rotary substep in the synthetic cycle of mammalian ATP synthase. The calculated free energy profiles capture a discrete pattern in the rotation of the central γ-shaft, with a metastable intermediate located-consistently with recent experimental findings-at 70° relative to the X-ray position. We identify this rotary step as the ATP-dependent substep, and find that the associated free energy input supports the mechanism involving concurrent nucleotide binding and release. During the main substep, our simulations show no significant opening of the ATP-bound β subunit; instead, we observe that mechanical energy is transmitted to its nucleotide binding site, thus lowering the affinity for ATP. Simultaneously, the empty subunit assumes a conformation that enables the enzyme to harness the free energy of ADP binding to drive ATP release. Finally, we show that ligand exchange is regulated by a checkpoint mechanism, an apparent prerequisite for high efficiency in protein nanomotors.

Insights into the origin of the high energy-conversion efficiency of F 1 -ATPase

“…By the rotation of γ to 80°with a binding FE of −9.8 kcal/mol, β E changes to β TP . This value corresponds to an FE change of −6.9 kcal/mol, a value very close to −6.7 kcal/mol determined by Czub et al (27) for the corresponding transition of an isolated αβ-dimer. The binding FE of ATP decreases further to −14.3 kcal/mol when β DP * is reached at 200°, where the catalytic dwell takes place and the bound ATP is hydrolyzed, as mentioned above.…”

“…Based on the chemical events in each substep, the 40°substep is referred to as the catalytic step and the 80°substep as the ATP binding step. A conformation first trapped by a molecular dynamics simulation (23), which is consistent with the structural changes suggested by single-molecule experiments (25,26), as well as a more recent simulation (27), is associated with the ATP binding "dwell." The open β E -conformation binds substrate ATP; the subsequent closing of β E induces a conformational change in an adjacent β-subunit to release the hydrolysis products of a previous ATP binding event, and together leads to the 80°substep of the γ-subunit.…”

“…We show here that a revision of the V-K&M model for the molecular coupled rotary-binding change mechanism, and the thermodynamic diagram of the enzyme cycle ( Fig. 1B) based on the atomistic simulation results (14,16,23,27,38), yield deeper insights into how F 1 -ATPase works. Fig.…”

“…3, ΔG o γ−rot ðθ i → θ f Þ refers to the FE change of the 80°γ-rotation. It may involve only the rotation of the globular portion of γ, while the changes in the coiled-coil portion of γ may occur with the α 3 β 3 -conformational change, caused by the contact interactions proposed by Pu and Karplus (16) and others (27,48,50).…”

Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity

“…Another success story comes from a study of L99A variant of bacteriophage T4 lysozyme where a range of metadynamics techniques was used not only to accurately capture key conformational and thermodynamic features, but also to uncover a transient tunnel that seems to allow ligands to unbind . Successful applications are not limited to metadynamics, as shown by a recent study that employed extensive umbrella sampling MD to describe the molecular details of the energy conversion mechanism of the main rotary step in the synthesis cycle of F 1 ‐ATPase . To achieve high efficiency and timely substrate binding and product release, this molecular motor requires tight coupling of its subunits.…”