Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Abstract

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F 1 -F o coupling

“…We observe an uneven distribution of particles between the three major rotary states, with 52% in state 1, 23% in state 2, and 21% in state 3, in line with other recent cryo-EM studies of ATP 3 of 10 (11)(12)(13) and with a recent singlemolecule study (20) that reported an asymmetry in the time-averaged population of rotary states in the presence of ADP. In comparing substates of a given primary rotary state, the later substates of the Polytomella complex (1D, 1E, 1F, 2C, 2D, and 3C) are consistently less populated than the earlier substates ( fig.…”

Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP

“…However, the proportion of particles corresponding to each reconstruction differed from our previous study (33% State 1, 44% State 2 and 23% State 3 in this study, compared with 46% State 1, 30% State 2 and 24% State 3 in Sobti et al, 2016), suggesting that a larger number of molecules were now observed in State 2 rather than State 1. Single molecule studies have also observed the enzyme populating the rotational dwell states in different proportions (Sielaff et al, 2019), suggesting that each dwell state may be at a different energy minimum. Inspection of the nucleotide-binding pockets in the α and β subunits revealed two further peaks, in addition to the four nucleotide peaks we reported previously for the autoinhibited enzyme (Sobti et al, 2016) (Figure 1—figure supplement 7).…”

pH-dependent 11° F1Fo ATP synthase sub-steps reveal insight into the Fo torque generating mechanism

“…The low, medium, and high efficiencies of TD formation reported here ( Fig 2B ) were attributed to torsional strain resulting from the asymmetry between 36° c 10 -ring stepping, and the 120° F 1 power strokes (8). Based on this asymmetry observed in low resolution ADP-inhibited F 1 F o structures (26), high efficiency TD formation was proposed to occur (27) in the rotary state comparable to that in which rotary sub-state structures PDB-IDs 6OQR and 6OQS were subsequently observed at 3.1 Å resolution (17). Sobti et al .…”