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High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na⁺-coupled ATP synthase.

Matthies D, Zhou W, Klyszejko AL, Anselmi C, Yildiz Ö, Brandt K, Müller V, Faraldo-Gómez JD, Meier T - Nat Commun (2014)

Bottom Line: To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution.Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit.These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany.

ABSTRACT
All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H(+) or Na(+). Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.

No MeSH data available.


Electrostatic barrier between the P and N-channels. (a) A snapshot was extracted from one of the simulations in which Na+ is spontaneously released (Fig. 5), and the exposed glutamate side-chain was paired to a guanidinium ion (GND+), modeled in to represent the interaction with the key arginine side-chain on TM4 of subunit-a. (b) The free-energy cost of transferring a bound Na+ from the adjacent site, counter-clockwise, to the site engaged to the GND+ ion, was then computed, by gradually decoupling the ion from its environment in configuration (a) and re-coupling it in configuration (b). (c) Free-energy change as a function of the (de)coupling parameter λ. The transfer free energy was calculated in both directions. The c-ring is represented as in Fig. 1. Side-chains and water molecules in the site are highlighted (sticks, spheres). Hydrogen atoms as well as other protein side-chains and all MPD/water molecules are omitted for clarity.
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Figure 7: Electrostatic barrier between the P and N-channels. (a) A snapshot was extracted from one of the simulations in which Na+ is spontaneously released (Fig. 5), and the exposed glutamate side-chain was paired to a guanidinium ion (GND+), modeled in to represent the interaction with the key arginine side-chain on TM4 of subunit-a. (b) The free-energy cost of transferring a bound Na+ from the adjacent site, counter-clockwise, to the site engaged to the GND+ ion, was then computed, by gradually decoupling the ion from its environment in configuration (a) and re-coupling it in configuration (b). (c) Free-energy change as a function of the (de)coupling parameter λ. The transfer free energy was calculated in both directions. The c-ring is represented as in Fig. 1. Side-chains and water molecules in the site are highlighted (sticks, spheres). Hydrogen atoms as well as other protein side-chains and all MPD/water molecules are omitted for clarity.

Mentions: To evaluate this key element of the mechanism, we calculated the free-energy cost associated with the transfer of a Na+ ion from a loaded binding site in the A. woodii c-ring (i.e. SP) to an adjacent empty site, clockwise (i.e. SN), in which the exposed carboxyl group is paired with a guanidinium ion (GND+), modeled in so as to mimic the interaction with the conserved arginine on subunit-a (Fig. 7a,b). The resulting free-energy value was ~9 kcal/mol (Fig. 7c). That is, an ion reaching SP through the P-channel would be more than a million times more likely to remain in SP than to hop to SN. Ion hopping would be also far less probable than the alternative next step in the proposed cycle, namely the rotational displacement of the c-ring (Fig. 6). The energy cost of this rotational displacement is about ~3 kcal/mol, i.e. three times the free energy cost of ATP synthesis at typical ATP/ADP/Pi concentrations (~11 kcal/mol), distributed over eleven steps. Direct ion hopping between the two c-ring binding sites exposed at the a/c interface is therefore extremely improbable, even from a purely thermodynamic standpoint (i.e. even if we neglect kinetic barriers). This electrostatic separation between the P- and N-channel ensures strict coupling between ion permeation and c-ring rotation.


High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na⁺-coupled ATP synthase.

Matthies D, Zhou W, Klyszejko AL, Anselmi C, Yildiz Ö, Brandt K, Müller V, Faraldo-Gómez JD, Meier T - Nat Commun (2014)

Electrostatic barrier between the P and N-channels. (a) A snapshot was extracted from one of the simulations in which Na+ is spontaneously released (Fig. 5), and the exposed glutamate side-chain was paired to a guanidinium ion (GND+), modeled in to represent the interaction with the key arginine side-chain on TM4 of subunit-a. (b) The free-energy cost of transferring a bound Na+ from the adjacent site, counter-clockwise, to the site engaged to the GND+ ion, was then computed, by gradually decoupling the ion from its environment in configuration (a) and re-coupling it in configuration (b). (c) Free-energy change as a function of the (de)coupling parameter λ. The transfer free energy was calculated in both directions. The c-ring is represented as in Fig. 1. Side-chains and water molecules in the site are highlighted (sticks, spheres). Hydrogen atoms as well as other protein side-chains and all MPD/water molecules are omitted for clarity.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: Electrostatic barrier between the P and N-channels. (a) A snapshot was extracted from one of the simulations in which Na+ is spontaneously released (Fig. 5), and the exposed glutamate side-chain was paired to a guanidinium ion (GND+), modeled in to represent the interaction with the key arginine side-chain on TM4 of subunit-a. (b) The free-energy cost of transferring a bound Na+ from the adjacent site, counter-clockwise, to the site engaged to the GND+ ion, was then computed, by gradually decoupling the ion from its environment in configuration (a) and re-coupling it in configuration (b). (c) Free-energy change as a function of the (de)coupling parameter λ. The transfer free energy was calculated in both directions. The c-ring is represented as in Fig. 1. Side-chains and water molecules in the site are highlighted (sticks, spheres). Hydrogen atoms as well as other protein side-chains and all MPD/water molecules are omitted for clarity.
Mentions: To evaluate this key element of the mechanism, we calculated the free-energy cost associated with the transfer of a Na+ ion from a loaded binding site in the A. woodii c-ring (i.e. SP) to an adjacent empty site, clockwise (i.e. SN), in which the exposed carboxyl group is paired with a guanidinium ion (GND+), modeled in so as to mimic the interaction with the conserved arginine on subunit-a (Fig. 7a,b). The resulting free-energy value was ~9 kcal/mol (Fig. 7c). That is, an ion reaching SP through the P-channel would be more than a million times more likely to remain in SP than to hop to SN. Ion hopping would be also far less probable than the alternative next step in the proposed cycle, namely the rotational displacement of the c-ring (Fig. 6). The energy cost of this rotational displacement is about ~3 kcal/mol, i.e. three times the free energy cost of ATP synthesis at typical ATP/ADP/Pi concentrations (~11 kcal/mol), distributed over eleven steps. Direct ion hopping between the two c-ring binding sites exposed at the a/c interface is therefore extremely improbable, even from a purely thermodynamic standpoint (i.e. even if we neglect kinetic barriers). This electrostatic separation between the P- and N-channel ensures strict coupling between ion permeation and c-ring rotation.

Bottom Line: To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution.Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit.These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany.

ABSTRACT
All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H(+) or Na(+). Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.

No MeSH data available.