Abstract
V1-ATPase (V1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis–derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V1 from the pathogen Enterococcus hirae (EhV1). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 107 m−1 s−1. At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, −80 and −40° backward steps were observed. The time constants of the pauses before −80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV1, including substeps largely different from those of F1-ATPases.
Highlights
V1-ATPase (V1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis– derived chemical energy into rotation
We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps
We previously reported that the EhV1 has two distinct reversible rotational states, namely clear and unclear, and concluded that the unclear rotation is caused by the unstable interaction between the stator A3B3 ring and the rotor DF subcomplex of the EhV1 [21, 55]
Summary
V-ATPase, Vacuolar ATPase; ATP␥S, adenosine 5Ј-(␥-thiotriphosphate); EhV-ATPase, Enterococcus hirae V-ATPase; V1, V1-ATPase; F1, F1-ATPase; EhV1, E. hirae V1; TtV1, T. thermophilus V1; TF1, thermophilic Bacillus PS3 F1; HF1, human mitochondrial F1; EF1, E. coli F1; YF1, yeast mitochondrial F1; BF1, bovine mitochondrial F1; KmATP, Michaelis constant of ATP-driven rotation; VmaxATP, maximum velocity of ATP-driven rotation; konATP, ATP-binding rate constant; 3D, three-dimensional; fps, frames per second; rps, revolutions per second; TEV, tobacco etch virus; PfGK, P. furiosus glucokinase; Ni-NTA, nickel-nitrilotriacetic acid. For F1, information of dynamics obtained by singlemolecule analysis, and high-resolution structures that correspond to elementary steps of the chemo-mechanical coupling cycle, have been obtained using F1 from bovine mitochondria (BF1) (36 –42), YF1 [43,44,45,46], EF1 [47, 48], and TF1 [49, 50]. The clear and unclear states were observed in EhV1 reconstituted from the isolated stator A3B3 ring and the rotor DF subcomplex, and in the recombinant A3B3DF complex, which has AviTag [57] in the D subunit for probe attachment. In the studies of EhV1, in addition to the single-molecule analysis of rotational dynamics, high-resolution structures with different conformations have been solved by X-ray crystallography [16, 17]. Constructed EhV1 with minimized additional amino acid residues showed only clear states and substeps in the rotation. From the results of detailed single-molecule analyses and the previous structural information [16, 17], here we propose a new model of a chemomechanical coupling scheme of EhV1 including substeps
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