Mechanism of Energy Conversion during the Rotary Catalytic Cycle of F1-ATPase

Abstract

FoF1-ATPase is a motor protein that synthesizes ATP from ADP and inorganic phosphate using the proton gradient across a membrane as a free energy source. When driven by a proton flux through the membrane-embedded Fo portion, the asymmetric gamma subunit of the F1 portion (gamma-shaft) undergoes a discrete rotation inside the cylinder of hexagonally arranged alpha and beta subunits. This causes the binding sites located at the beta subunits to cycle between states of different affinity for nucleotides and leads to the synthesis of ATP against the chemical potential gradient.To examine the process of mechanochemical energy conversion during the rotary synthesis, we determined the free energy profile for the rotation of the gamma-shaft within the alpha3beta3 catalytic headpiece cylinder by 120 degrees, starting from its angular position captured in typical x-ray structures. To this end, we used atomistic molecular dynamics and umbrella sampling with the set of initial configurations obtained from the flexible-axis rotation simulation, in which the gamma-shaft was driven to rotate for 1 microsecond from the initial position with its convex side facing the empty subunit (0 degrees) to the final position facing the nucleotide-bound subunit (120 degrees). With this approach we found the presence of a second minimum of the free energy likely corresponding to the so-called ATP-dependent pause of F1-ATPase. By decomposing the obtained free energy profile into contributions due to individual interactions, we identified the major determinants of the stability of this state. Furter, by relating the rotor angular position to the catalytic headpiece conformational state and by applying force decomposiion analysis, we thoroughly analyzed the pathways of energy transfer between the rotating gamma-shaft and the binding sites.