Changes within the central stalk of E. coli F1Fo ATP synthase observed after addition of ATP

Abstract

F₁F_o ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the F_o motor that is transferred to the F₁ motor to catalyze ATP production, with flexible F₁/F_o coupling required for efficient catalysis. F₁F_o ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F₁F_o ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the F_ο motor. This highlights the importance of the central rotor for flexible coupling of the F₁ and F_o motors and provides further insight into the regulatory mechanism mediated by subunit ε.

Fig. 1. The ε subunit of E. coli F₁F_o ATP synthase.

a Schematic of E. coli F₁F_o ATP synthase with subunits colored and labeled. The ε subunit is shown in green in the inhibited up conformation. b Crystal structure of the ε subunit in the up conformation (PDB:3oaa), with α, β, and γ subunits removed for clarity. c Crystal structure of the isolated ε subunit in the down conformation (PDB:1aqt).

Fig. 2. Nucleotide occupancy and conformational changes in the F₁ motor following incubation with MgATP.

a Horizontal section of the State 2 E. coli F₁F_o ATP synthase cryo-EM map and details of the β subunit catalytic site occupancies (with equivalent mitochondrial F₁ nomenclature: β1 = β_DP, β2  =  β_E, β3 = β_TP, as named for the E. coli enzyme). β1 contains MgADP, β2 contains ADP, and β3 contains MgATP. Section of map contoured to 0.028 in ChimeraX and mesh for nucleotides contoured to isolevel 10 in PyMol (Schrödinger). b, c Comparison of the F₁ motor after incubation with MgATP (this study; γ in blue, ε in green and β in yellow) or MgADP (PDB:6oqv; subunits shown as outline). b The central stalk (subunits γ and ε) is rotated ~10° clockwise when viewed from the membrane (structures are aligned to the F₁ β barrel crown). c β1 closes inwards from a half-open to closed conformation.

Fig. 3. E. coli F₁F_o ATP synthase ε subunit in three conformational sub-states.

Cryo-EM maps (transparent surface) and molecular models (cartoon representation) of the E. coli F₁F_o ATP synthase rotor in three conformation sub-states. Subunit γ in light blue and ε in green, with the foot helix of subunit γ labeled in dark blue. a The εCTD up sub-state observed after addition of 10 mM MgADP (PDB:6oqv; EMDB:20171). b The εCTD half-up sub-state observed after addition of 10 mM MgATP (State 2 half-up in this study). c The εCTD down sub-state observed after addition of 10 mM MgATP (State 2 down in this study). See Supplementary Fig. 5 for close up views of the cryo-EM maps for the εCTD and γ foot.

Fig. 4. Structural rearrangements of E. coli F₁F_o ATP synthase between the half-up and down conformations.

Comparison of State 2 εCTD half-up conformation (top panels) with State 2 εCTD down conformation (bottom panels). a Overall view describing the εCTD (green) transition and rotation of the c-ring (gray with one c subunit colored black). b When superposed on subunit a (orange), the c-ring rotates two c subunits in the F_o motor (one c subunit colored black with black arrow—relative rotation of c subunits identified using the interaction between εNTD and c subunit; Supplementary Fig. 6). c When superposed on the β barrel crown of the α and β subunits, the εNTD rotates ~50° about the γ subunit and the peripheral stalk flexes ~15°. d When superposed on N-terminus of subunit γ, the foot helix (residues γ39-57) bends and twists to accommodate the movement of the εNTD.

Fig. 5. Movements of the central and peripheral stalks.

Side views showing the comparison of State 2 half-up and down sub-states. a Intact F₁F_o complexes shown as tube cartoon. b The peripheral stalk (dimer of b subunits) bends counterclockwise and c the central stalk twists clockwise, facilitating a rotation of two c subunits in the F_o motor.

Fig. 6. Rotation of the εNTD increases the distance to the εCTH1 binding site.

To highlight the relative rotation of the ε subunit between the half-up and down sub-states, the State 2 half-up ε subunit was superposed onto the State 2 down εNTD (shown in yellow). After the εNTD rotates about the γ subunit, the distance between εCTH1 and its binding site on γ is increased in the down sub-state and is less likely to attach to the central stalk.

Fig. 7. Subunit ε attenuates ATPase activity.

a εΔCTH2 truncation retains the εCTH1 which can bind to the γ subunit. b εΔCTH1 + 2 retains only the εNTD akin to the εCTD down conformation. c Cryo-EM maps of the 3 states generated using the εΔCTH2 truncation mutant after exposure to 10 mM MgATP showed E. coli F₁F_o ATP synthase in the half-up sub-state, with εCTH1 attached to the subunit γ. The 3 states have been rotated successively by 120° to show the position of subunit ε. d Coomassie stained SDS PAGE (uncropped image provided as Supplementary Data 5) of purified E. coli F₁F_o ATP synthase WT, εΔCTH2 and εΔCTH1 + 2. Subunits labeled and minor contamination bands identified as; (i) Ribonuclease E, (ii) GroEL, and (iii) ElaB. e ATP regeneration assays of WT (containing full length subunit ε), εΔCTH2 (ε1–104) and εΔCTH1 + 2 (ε1–81). All data points and mean are shown (raw traces are in Supplementary Fig. 8 and values in Supplementary Data 6). Removal of εCTH2 results in higher ATP turnover than WT. Removal of both εCTH1 and εCTH2 shows higher ATP turnover than removal of only εCTH2.

Fig. 8. Rotation in the F_o motor in the half-up and down sub-states.

a The F_o rotor of the State 2 down sub-state trails the State 2 half-up sub-state by two c subunit steps in the synthesis direction. b When the F_o motor is driven in the synthesis direction, the central stalk is unclamped and the εNTD is pushed away from the εCTH1 binding site on γ, reducing the likelihood of the up conformation. c When the F_o motor is driven in the reverse direction, the central stalk is closed and the εNTD is pushed towards the εCTH1 binding site on γ, increasing the likelihood of the up conformation.

Fig. 9. Schematic of clamping by εCTH1.

When the εCTH1 is in the half-up state and bound to subunit γ, the central stalk is clamped and increases the stiffness of the rotor. When εCTH1 is in the down state, the central stalk is no longer clamped and has increased flexibility.