Mechanism of ATP hydrolysis dependent rotation of bacterial ATP synthase

Abstract

F₁ domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α₃β₃ fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αβ dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F₁ domain in F_oF₁ synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F₁ domain when nucleotides are bound at all the three catalytic αβ dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at α_Dβ_D, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

Fig. 1. Structures and rotation mechanism of F_oF₁.

a Cryo-EM structure of F_oF₁ ATP synthase in the step-waiting conformation. Each subunit is colored differently. The F₁ domain contains three catalytic αβ dimers which surround the γ subunit. b, c Side views (upper) and bottom section views (lower) of the F₁ domain in the step-waiting (0°) and 81° structure, respectively. The three catalytic dimers are represented different colors: α_E (marine blue) and β_E (light blue), α_T (moss green) and β_T (light green), and α_D (purple) and β_D (light purple). The β_D subunit in step-waiting adopts a more open structure, termed as DHO. The γ subunits are represented as a gray tube in the center of both α₃β₃ sub-complexes. d A proposed scheme for chemo-mechanical coupling during a 120° rotation step of the F₁ domain driven by ATP. In this model, ATP binding to the F₁ domain immediately initiates the 80° rotation with an associated release of ADP. ATP is hydrolyzed at the 80° dwell position with no associated rotation of γ. It is the release of Pi from the enzyme which is suggested to drive the final 40° rotation.

Fig. 2. Structure of 81° (left), post-hyd (center), and 101°(right) at high [ATP].

a Cross section of the F₁ domain showing the catalytic sites viewed from the F_o side. Each catalytic dimer is shown in ribbon representation and colored as detailed in Fig. 1. The bound nucleotides are represented as spheres. b Side view structure of α_Dβ_D dimer. The left panel is a magnified view of the catalytic interface at each α_Dβ_D. c Structure of the catalytic site in α_Dβ_D. Amino acid residues and bound nucleotide and Pi are represented as sticks. Lower panels; Stick representation model of ATP/ADP and Pi with EM density in α_Dβ_D. The distance between γ and β phosphate of the ATP in each conformational state is shown in blue (Å). Each distance was calculated using Chimera software. d Structure of the catalytic site in α_Eβ_E. The EM density of ATP / Pi is superimposed onto the model. The distance between Pi and γ phosphate of ATP is shown in yellow (Å).

Fig. 3. Structure comparison of 6 intermediates captured during the 40° step at high [ATP].

a Cross section of the F₁ domain showing the catalytic site. 81°, post-hyd, 91°, 101°, and 120° structures are arranged from left to right. The Cα displacement relative to the next structure (right side) is indicated by the red-white color gradient. The dashed square indicates the area of this figure shown in the zoomed in view in b–f. b–f Comparison of each structure to the following one. b compares the γ subunit and its surroundings, and the different rotation angles between each γ subunit and that of the next structure are indicated by different colored lines. c shows an upper view of β_D, d compares β_D from a side view, and e, f compare α_T (120° and step waiting) and α_E (91° and 101°), respectively. Each cartoon chain is colored as 81°(gray), 83°(green), 91°(blue), 101°(pink), 120°(light green), and step-waiting (orange). g Structures of the catalytic site of 91° in α_Eβ_E. The key side chains involved in coordinating Pi and nucleotide are shown in stick representation. Semi-transparent electron density for the Pi and nucleotide is shown over the stick representations. h Superimposition of the side chains of α_Eβ_E in 91°(orange) with those of α_Eβ_E in 101° (light blue).

Fig. 4. Structures captured at low [ATP].

a Cross section of the F₁ domain in 81° showing the catalytic sites viewed from the F_o side. The bound nucleotides are represented as spheres. The electron density of the nucleotides within the enclosed circles are shown in larger view on the right. The distance between γ (or Pi) and β phosphate of the ATP in each conformational state is shown in blue (Å). Each distance was calculated using Chimera software. b Cross section of the F₁ domain in ATP-waiting (120°). c Comparison of the Pi binding site in α_Eβ_E in the 91° and 120° structures with electron density indicated for the Pi. d Comparison of the catalytic site of the α_Eβ_E in ATP waiting (cream) and step waiting (purple). ATP in step waiting is represented by the orange and blue sticks. The step waiting structure was captured at high [ATP].

Fig. 5. ATP driven rotation scheme of F_oF₁.

a Under low [ATP] conditions, the catalytic site in α_Eβ_E of ATP waiting remains empty. b The step-waiting is formed by binding of ATP to α_Eβ_E of ATP waiting. c The step-waiting initiates the 80° rotation step of the γ subunit coupled with structure transition of the three αβ dimers; α_Eβ_E to α_Tβ_T with a zippering motion caused by binding of ATP to α_Eβ_E, α_Tβ_T to α_Dβ_D, and α_Dβ_D^HO to α_Eβ_E with associated release of ADP via an unzippering motion of α_Dβ_D^HO. d ATP bound to α_Tβ_T is hydrolyzed in the α_Dβ_D dimer of 81° just after the 81° rotation. e, f Once ATP bound to α_Dβ_D is hydrolyzed, an unzippering motion of α_Dβ_D (purple arrows) proceeds via a 10° rotation step of γ, resulting in the structural change of hydrolysable to 91° through post-hyd. The outward motion of α_E in 91°(light blue arrow) in concert with a further 10° rotation induces release of Pi, resulting in 101°which adopts a more open α_Eβ_E. g The final rotation from 101° to 120° occurs without structural change in any of three catalytic dimers. h Further motion of the CT domain of α_E induces the structural between 120° and, i ATP waiting without any associated rotation of the γ subunit. j The step-waiting (120°) is formed by binding of ATP to α_Eβ_E of ATP waiting. The α_Eβ_E of six intermediates (81°, post-hyd, 91°, 101°, 120°, and ATP waiting) are occupied with ATP at high [ATP], indicating that ATP binds to empty α_Eβ_E at any rotation angle of γ (light blue dash arrows). k 360° rotation of F_oF₁. CryoEM maps of F_oF₁ obtained at high [ATP] are placed in a circular arrangement according to the angle of the γ subunit. The three state maps are represented by yellow (state1), blue (state2), and green (state3), respectively.