Anatomy of F 1 -ATPase powered rotation

The rotary motor enzyme F o F 1 -ATP synthase uses the protonmotive force across a membrane to synthesize ATP from ADP and P i (H 2 PO 4 − ) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 10 5 . This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F 1 -ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40°from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why P i is not released immediately after ATP hydrolysis, but only after a subsequent 120°rotation, in agreement with experiment. A concerted conformational change of the α 3 β 3 crown is shown to induce the 40°rotation of the γ-subunit only when the β E subunit is empty, whereas with P i bound, β E serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F 1 -ATPase achieves the coupling between the small changes in the active site of β DP and the 40°rotation of γ. T he molecular motor F o F 1 -ATP synthase is composed of two domains: a transmembrane portion (F o ), the rotation of which is induced by a proton gradient, and a globular catalytic moiety (F 1 ) that synthesizes and hydrolyzes ATP. The primary function of the proton-motive force acting on F o F 1 -ATP synthase is to provide the torque required to rotate the γ-subunit in the direction for ATP synthesis (1, 2). The catalytic moiety, F 1 -ATPase, has an α 3 β 3 "crown" composed of three α-and three β-subunits arranged in alternation around the γ-subunit, which has a globular base and an extended coiled-coil portion (3) (Fig. 1A). F 1 -ATPase by itself binds ATP and hydrolyzes it to induce rotation of the γ-subunit (in the opposite direction from that for synthesis) on the millisecond time scale under optimum conditions (4, 5). All of the α-and β-subunits bind nucleotides, but only the three β-subunits are catalytically active. The original crystal structure (3) of F 1 -ATPase from bovine heart mitochondria (MF 1 ) led to the identification of three conformations of the β-subunit: β E (empty), β TP (ATP analog bound), and β DP (ADP bound); Fig. 1A. In the known structures of F 1 -ATPase, which apparently are near the "catalytic dwell" state, the state in which catalysis occurs (6, 7), the β E subunit conformation is partly to fully open and is very different from those of the β TP and β DP subunits, which are closed and very similar to each other (SI Appendix, SI1). Searching for the ATP Waiting StateBecause no X-ray structure is available for the ATP waiting state, we searched for it by molecular dynamics (MD) simulations with an external torque applied to the γ-subunit in t...

Section: Timing Of P I Releasementioning

confidence: 70%

Trapping the ATP binding state leads to a detailed understanding of the F ₁ -ATPase mechanism

Nam

Karplus

2014

“…The time scale with the three substeps (∼40 ms) agrees well with the experimental values (∼50 ms; ∼60 s −1 for a 120°step) at a maximum load condition (4). This result suggests a possible third substep, yet unresolved, for the E. coli F 1 (34,35). This third step could be associated with phosphate release (31,36).…”

Section: Significancementioning

confidence: 91%

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...

“…The intact molecular motor has a mass of 525 kDa. F o F 1 investigations are typically conducted under hydrolysis conditions (1,6,7), where γec 10 rotation is driven by the β subunits that cycle through a series of conformations (8). ATP hydrolysis triggers power strokes that involve consecutive interactions of the β-levers with γ (3, 6, 9).…”

mentioning

confidence: 99%

“…ii) Reverse rotation during dwell periods: Individual power strokes cause γ to turn 120°in a clockwise direction, when viewed from the top of the α 3 β 3 crown (40). Each power stroke is followed by a dwell, during which none of the β-levers actively drives γ rotation (6). One may contemplate whether PMF can cause reverse (counterclockwise) rotation of c 10 by a small angle (<<120°) during these dwells, as suggested by some early experiments (41).…”

mentioning

confidence: 99%

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

Vahidi

Dunn

et al. 2016

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.