Viscoelastic Dynamics of Actin Filaments Coupled to Rotary F-ATPase: Angular Torque Profile of the Enzyme

127

F 1 -ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120°power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35°from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85°to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85°of rotation, consistent with a transition from F 1 structures 2HLD1 and 1H8E (Protein Data Bank).

Section: Significancementioning

confidence: 99%

Anatomy of F ₁ -ATPase powered rotation

Martin

Ishmukhametov

Hornung

et al. 2014

127

“…We account for the torsional elasticity and friction by describing the rotational motion of the γ-subunit as overdamped Langevin dynamics on a 2D harmonic free energy surface. The model quantifies the magnitude of transient elastic energy storage compensating for the incommensurate rotational symmetries of the F o and F 1 motors (30). The resulting energetic constraints allow us to map out a detailed pathway for their coupled rotary motions, and to rationalize the finer stepping of the mammalian F 1 motor seen in recent experiments (31), with only eight c subunits in the corresponding F o motor.…”

mentioning

confidence: 99%

Elasticity, friction, and pathway of γ-subunit rotation in F _o F ₁ -ATP synthase

Okazaki

Hummer

2015

We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of F o F 1 -ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torquedriven γ-subunit rotation in the F 1 -ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than k B T per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the F o and F 1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F 1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in F o and F 1 , respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.bioenergetics | molecular motor | mechanochemical coupling F o F 1 -ATP synthase is essential for life. From bacteria to human, this protein synthesizes ATP from ADP and inorganic phosphate P i in its F 1 domain, powered by an electrochemical proton gradient that drives the rotation of its membrane-embedded F o domain (1-5). Its two rotary motors, F 1 and F o , are coupled through the γ-subunit forming their central shaft (2). ATP synthase is a fully reversible motor, in which the rotational direction switches according to different sources of energy (2, 6). In hydrolysis mode, the F 1 motor pumps protons against an electrochemical gradient across the membrane-embedded F o part, converting ATP to ADP and P i (7,8).F 1 has a symmetric ring structure composed of three αβ-subunits with the asymmetric γ-subunit sitting inside the ring (9, 10). Each αβ-subunit has a catalytic site located at the αβ-domain interface. The F 1 ring has a pseudothreefold symmetry with the three αβ-subunits taking three different conformations, E (empty), TP (ATP-bound), and DP (ADP bound) (9-11). The F o part is composed of a c ring and an a subunit (3, 12). Driven by protons passing through the interface of the c ring and the a subunit, the c ring rotates together with the γ-subunit (rotor) relative to the a subunit, which is connected to the F 1 ring through the peripheral stalk of the b subunit (stator) (12). Interestingly, in nature, one finds a large variation in the number of subunits in the c ring. In animal mitochondria, one finds c 8 rings, requiring a minimal number of e...

“…In the simplest version of bacterial F o F 1 such as F o F 1 s from thermophilic Bacillus PS3 and Escherichia coli, subunit compositions of F 1 and F o are, respectively, α 3 β 3 γδe and ab 2 c 10 . Both portions are rotary motors that share a common rotor shaft γec 10 . Downward proton flow through F o along the gradient of the electrochemical potential of the proton across the membrane drives the rotation of the c 10 rotor ring in F o that drags rotation of the γe rotor shaft of F 1 in the surrounding α 3 β 3 cylinder.…”

mentioning

confidence: 99%

“…The torque of this motor has been estimated with various methods (8)(9)(10)(11)(12)(13). Among them, an early study that used counter torque reported the torque was ∼50 pN nm/rad (10), indicating that efficiency in chemomechanical energy conversion by F 1 from chemical energy of ATP hydrolysis (ΔG ATP ) to mechanical work of rotation reaches almost 100%.…”

mentioning

confidence: 99%

Simple mechanism whereby the F ₁ -ATPase motor rotates with near-perfect chemomechanical energy conversion

Saita

Suzuki

Kinosita

et al. 2015

F 1 -ATPase is a motor enzyme in which a central shaft γ subunit rotates 120°per ATP in the cylinder made of α 3 β 3 subunits. During rotation, the chemical energy of ATP hydrolysis (ΔG ATP ) is converted almost entirely into mechanical work by an elusive mechanism. We measured the force for rotation (torque) under various ΔG ATP conditions as a function of rotation angles of the γ subunit with quasistatic, single-molecule manipulation and estimated mechanical work (torque × traveled angle) from the area of the function. The torque functions show three sawtooth-like repeats of a steep jump and linear descent in one catalytic turnover, indicating a simple physical model in which the motor is driven by three springs aligned along a 120°rotation angle. Although the second spring is unaffected by ΔG ATP , activation of the first spring (timing of the torque jump) delays at low [ATP] (or high [ADP]) and activation of the third spring delays at high [P i ]. These shifts decrease the size and area of the sawtooth (magnitude of the work). Thus, F 1 -ATPase responds to the change of ΔG ATP by shifting the torque jump timing and uses ΔG ATP for the mechanical work with near-perfect efficiency. T he F o F 1 -ATP synthase (F o F 1 ) is a ubiquitous enzyme located in bacterial plasma membranes, mitochondrial inner membranes, and chloroplast thylakoid membranes. It plays a critical role in energy metabolism by synthesizing ATP from ADP and inorganic phosphate (P i ). This enzyme consists of and is separable into two major portions: membrane-embedded F o and water-soluble F 1 . In the simplest version of bacterial F o F 1 such as F o F 1 s from thermophilic Bacillus PS3 and Escherichia coli, subunit compositions of F 1 and F o are, respectively, α 3 β 3 γδe and ab 2 c 10 . Both portions are rotary motors that share a common rotor shaft γec 10 . Downward proton flow through F o along the gradient of the electrochemical potential of the proton across the membrane drives the rotation of the c 10 rotor ring in F o that drags rotation of the γe rotor shaft of F 1 in the surrounding α 3 β 3 cylinder. This rotation causes cyclic conformational changes in each of the three catalytic β subunits that result in ATP synthesis (1-3).The isolated F 1 , often called F 1 -ATPase, catalyzes the ATP hydrolysis reaction that drives the rotation of γe to the direction opposite to that in the ATP synthesis. The minimum subunit composition as an ATPase rotary motor is α 3 β 3 γ, and we refer to this complex hereinafter as F 1 . The γ rotates 120°per net hydrolysis of one ATP. Extensive studies, mainly on F 1 from thermophilic Bacillus PS3, have established the nearly complete catalytic scheme of the ATP-driven rotation: Starting from the ATP-waiting state where the orientation angle of the γ is set as 0°, ATP binding to the first β induces the 80°-step rotation of the γ, ADP-release from the second β occurs at some point during this step rotation, and the previously bound ATP in the third β is hydrolyzed at 80°. Then P i release from either the se...