The ATP-waiting conformation of rotating F
            <sub>1</sub>
            -ATPase revealed by single-pair fluorescence resonance energy transfer

Yasuda, Ryohei; Masaike, Tomoko; Adachi, K.; Noji, Hiroyuki; Itoh, Hiroyasu; Kinosita, Kazuhiko

doi:10.1073/pnas.1637860100

Cited by 93 publications

(92 citation statements)

References 31 publications

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“…2B). These nucleotide conditions are thought to mimic the ATP-bound state (13,22), the ADP-inhibited state (14), and the catalytic intermediate (15), respectively, and stabilize the 80°position. Therefore, it is likely that transition of ␣-helices of the ⑀ subunit from the extended form to the retracted form can take place when the ␥ subunit in F 1 dwells at the 80°position.…”

Section: Discussionmentioning

confidence: 99%

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Iino

Murakami

Iizuka³

et al. 2005

Journal of Biological Chemistry

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It has been proposed that C-terminal two ␣-helices of the ⑀ subunit of F 1 -ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F 1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the ␥ subunit and C terminus of the ⑀ subunit, respectively. High FRET (extended form) of F 1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5-Adenyl-␤,␥-imidodiphosphate, ADP ؉ AlF 4 ؊ , ADP ؉ NaN 3 , and GTP also caused the retracted form, indicating that ATP binding to the catalytic ␤ subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ⑀ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65°C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ⑀ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65°C reached to 0.67 mM, a consistent value to assume that the ⑀ subunit acts as a sensor of ATP concentration in the cell.A rotary motor F 1 -ATPase (F 1 ) 2 is a water-soluble portion of F 0 F 1 -ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (1, 2). F 1 has a subunit structure of ␣ 3 ␤ 3 ␥␦⑀; ␣ and ␤ subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; ␥ subunit rotates in the ␣ 3 ␤ 3 ring; ␦ subunit connects the ring to the stator part of F 0 ; and ⑀ subunit rotates together with ␥ subunit as a body. The ⑀ subunit (ϳ14 kDa) has a regulatory function and consists of N-terminal ␤-sandwitch and C-terminal two ␣-helices (3, 4).Previous structural studies of F 1 indicated two conformations of the ⑀ subunit with different arrangement of the two ␣-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (5-8). Cross-linking studies suggested the third conformation with fully extended ␣-helices 3 (Fig. 1C) (9). Biochemical data have indicated that the ⑀ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a noninhibitory conformation (9). Thus, it appears that the regulatory function of the ⑀ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work. Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational...

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Section: Discussionmentioning

confidence: 99%

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Iino

Murakami

Iizuka³

et al. 2005

Journal of Biological Chemistry

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“…(i) A FRET experiment suggests that the 1994 structure corresponds to the one formed after a 90°substep (38). (ii) A structural change in ␤ upon phosphate release has been reported (39).…”

Section: Fig 4 Residues That Transfer the Torque According To The mentioning

confidence: 99%

Folding-based molecular simulations reveal mechanisms of the rotary motor F ₁ –ATPase

Koga

Takada

2006

Proc. Natl. Acad. Sci. U.S.A.

120

146

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Biomolecular machines fulfill their function through large conformational changes that typically occur on the millisecond time scale or longer. Conventional atomistic simulations can only reach microseconds at the moment. Here, extending the minimalist model developed for protein folding, we propose the ''switching Go model'' and use it to simulate the rotary motion of ATP-driven molecular motor F 1-ATPase. The simulation recovers the unidirectional 120°rotation of the ␥-subunit, the rotor. The rotation was induced solely by steric repulsion from the ␣3␤3 subunits, the stator, which undergoes conformation changes during ATP hydrolysis. In silico alanine mutagenesis further elucidated which residues play specific roles in the rotation. Finally, regarding the mechanochemical coupling scheme, we found that the tri-site model does not lead to successful rotation but that the alwaysbi-site model produces Ϸ30°and Ϸ90°substeps, perfectly in accord with experiments. In the always-bi-site model, the number of sites occupied by nucleotides is always two during the hydrolysis cycle. This study opens up an avenue of simulating functional dynamics of huge biomolecules that occur on the millisecond time scales involving large-amplitude conformational change.always-bi-site model ͉ energy landscape ͉ funnel ͉ switching Go model B iomolecular machines, such as the ribosome, transporter, and molecular motors, fulfill their function through largeamplitude conformational change. Structural information before and after the conformational change has been provided by x-ray crystallography and other methods for many cases (1). However, these methods do not directly observe the molecular dynamics that connects two-end structures. These dynamical aspects can be observed directly by fluorescence and other time-resolved spectroscopy; however, the latter methods monitor local structure but do not give global structural information. In this sense, molecular dynamics simulation is potentially powerful because it can provide full time-dependent structural information about biomolecular machines (2-5). Yet, functional cycles of these systems typically take milliseconds or longer, which is far beyond the current reach of molecular simulations with all-atom standard force-fields (6). Thus, a complementary approach may be to coarse-grain the molecular representation, thereby enabling the simulation orders of magnitude-longer time scales.Coarse-graining drops some details from the model, while hopefully keeping the essence. To achieve them, we need some perspective on the physical process we are trying to simply. Upon conformational change, some portions of molecules do not significantly change their structures; here, the atomic motion can be well approximated by harmonic fluctuations. It has been known that very simple C ␣ models, such as the elastic network model (7), can reproduce low-frequency harmonic fluctuation fairly well. Conversely, large-scale conformational change involves rearrangement of nonlocal contacts between amino acids, which is...

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“…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).…”

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confidence: 99%

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

Nam

Karplus

2014

Proc. Natl. Acad. Sci. U.S.A.

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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...

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The ATP-waiting conformation of rotating F ₁ -ATPase revealed by single-pair fluorescence resonance energy transfer

Cited by 93 publications

References 31 publications

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Folding-based molecular simulations reveal mechanisms of the rotary motor F ₁ –ATPase

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

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The ATP-waiting conformation of rotating F 1 -ATPase revealed by single-pair fluorescence resonance energy transfer

Cited by 93 publications

References 31 publications

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Folding-based molecular simulations reveal mechanisms of the rotary motor F 1 –ATPase

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

Contact Info

Product

Resources

About

The ATP-waiting conformation of rotating F ₁ -ATPase revealed by single-pair fluorescence resonance energy transfer

Folding-based molecular simulations reveal mechanisms of the rotary motor F ₁ –ATPase

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