Correlation between the conformational states of F
            <sub>1</sub>
            -ATPase as determined from its crystal structure and single-molecule rotation

The molecular description of the mechanism of F 1 -ATPase is based mainly on high-resolution structures of the enzyme from mitochondria, coupled with direct observations of rotation in bacterial enzymes. During hydrolysis of ATP, the rotor turns counterclockwise (as viewed from the membrane domain of the intact enzyme) in 120°steps. Because the rotor is asymmetric, at any moment the three catalytic sites are at different points in the catalytic cycle. In a "ground-state" structure of the bovine enzyme, one site (β E ) is devoid of nucleotide and represents a state that has released the products of ATP hydrolysis. A second site (β TP ) has bound the substrate, magnesium. ATP, in a precatalytic state, and in the third site (β DP ), the substrate is about to undergo hydrolysis. Three successive 120°turns of the rotor interconvert the sites through these three states, hydrolyzing three ATP molecules, releasing the products and leaving the enzyme with two bound nucleotides. A transition-state analog structure, F 1 -TS, displays intermediate states between those observed in the ground state. For example, in the β DP -site of F 1 -TS, the terminal phosphate of an ATP molecule is undergoing in-line nucleophilic attack by a water molecule. As described here, we have captured another intermediate in the catalytic cycle, which helps to define the order of substrate release. In this structure, the β E -site is occupied by the product ADP, but without a magnesium ion or phosphate, providing evidence that the nucleotide is the last of the products of ATP hydrolysis to be released.catalytic mechanism | magnesium release | nucleotide release H igh-resolution structures of F 1 -ATPase from bovine heart and yeast mitochondria have provided the molecular basis for the catalytic mechanism of ATP synthesis and hydrolysis by F 1 F oATPase (1-10). During ATP synthesis, a mechanical rotation of the γ-subunit driven by the transmembrane proton-motive force, exerted on a ring of C-subunits in the F o membrane domain of the intact enzyme, takes each of the three β-subunits in its catalytic domain through the states represented by the β TP -, β DP -, and β Esubunits, thereby synthesizing three ATP molecules during each 360°rotation (1, 11). Hydrolysis of ATP reverses the direction of rotation, and protons are ejected from the mitochondrial matrix into the intermembrane space through the F o domain of the enzyme (12, 13). The rotation of the γ-subunit is not continuous, but it proceeds in 120°steps consisting of 90°and 30°substeps (14). The ground-state structure of F 1 -ATPase (denoted F 1 -GS) probably represents the state of the enzyme at the end of the complete 120°rotary step.Attempts have been made to access structures that represent conformations of β-subunits that are intermediate between the three conformations in the ground-state structure by inhibiting the enzyme in various ways. However, many of the inhibitors arrest the catalytic cycle in the ground state (15-17), except that the natural inhibitor protein IF 1 arrests bovine F ...

Section: Resultsmentioning

confidence: 99%

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria

Rees

Montgomery

Leslie

et al. 2012

“…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).…”

mentioning

confidence: 99%

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

Nam

Karplus

2014

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...

“…However, we still do not have a clear structure function correlation that involves both the chemistry and the conformational coupling. The challenge became even more exciting in view of the remarkable progress in single-molecule studies that directly visualized the γ-subunit rotation in a unidirectional way, while revealing several plausible intermediates that have not been observed in any of the crystal structures (9)(10)(11).…”

mentioning

confidence: 99%

“…For example, despite the fact that the structures of several ATPases have been elucidated (e.g., refs. 4 and 8) and recent advances in detailed elucidation of some key steps have been made (9)(10)(11), it is not completely clear at what stage in the reaction energy is actually released and, in turn, what is the nature of the energy conversion process. Some workers have suggested that the major source of the energy release is the conversion of the free energy of ATP binding into elastic strain, which is subsequently released by a coordinated and tightly coupled conformational mechanism (6).…”

mentioning

confidence: 99%

Electrostatic origin of the mechanochemical rotary mechanism and the catalytic dwell of F1-ATPase

Mukherjee

Warshel

2011

102

152

Understanding the nature of energy transduction in life processes requires a quantitative description of the energetics of the conversion of ATP to ADP by ATPases. Previous attempts to do so have provided an interesting insight but could not account for the rotary mechanism by a nonphenomenological structure/energy description. In particular it has been very challenging to account for the observations of the 80°and 40°rotational substates, without any prior information about such states in the simulation procedure. Here we use a coarse-grained model of F1-ATPase and generate, without the adjustment of phenomenological parameters, a structure-based free energy landscape that reproduces the energetics of the mechanochemical process. It is found that the landscape along the relevant rotary path is determined by the electrostatic free energy and not by steric effects. Furthermore, the generated surface and the corresponding Langevin dynamics simulations identify a hidden conformational barrier that provides a new fundamental interpretation of the catalytic dwell and illuminate the nature of the energy conversion process.bioenergetics | chemical-conformational coupling | molecular motors | coarse grain model U nderstanding the energetics of the biological conversion of ATP to ADP is crucial for elucidating the nature of energy transduction in life processes and also for practical understanding of the action of molecular motors (1, 2). At present it seems that the detailed nature of this biological energy conversion process has remained a major puzzle (1-7). For example, despite the fact that the structures of several ATPases have been elucidated (e.g., refs. 4 and 8) and recent advances in detailed elucidation of some key steps have been made (9-11), it is not completely clear at what stage in the reaction energy is actually released and, in turn, what is the nature of the energy conversion process. Some workers have suggested that the major source of the energy release is the conversion of the free energy of ATP binding into elastic strain, which is subsequently released by a coordinated and tightly coupled conformational mechanism (6). It was also postulated that the energy conversion involves an inertial energy release (12), whereas our works (13, 14) have suggested that the energetics is associated with the electrostatic work, and the origin of the free energy change involves a significant contribution from the charge separation in solution outside the ATPase active sites. Furthermore, biochemical and structural studies have provided a molecular model for the action of ATPases (1-4), and remarkable phenomenological analyses (6, 7) indicated what are the conditions for effective action. However, we still do not have a clear structure function correlation that involves both the chemistry and the conformational coupling. The challenge became even more exciting in view of the remarkable progress in single-molecule studies that directly visualized the γ-subunit rotation in a unidirectional way, while revealing several...