Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase

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.

Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking

“…In this study, by using scattering imaging of gold nanoparticle (AuNP), we visualized load-free fast stepping motion of artificially-dimerized chimeric dynein with 100 µs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. Since AuNP strongly scatters light at its plasmon resonance wavelength without suffering from photobleaching and blinking, it has been used as a probe of single-molecule imaging of linear and rotary motor proteins in vitro [17][18][19][20][21][22][23][24][25] and imaging of intracellular cargo transport [26][27][28] . We analyzed trajectory of the stepping motion of the chimeric dynein in detail, including the step size, preference of the step direction, and dwell time, and revealed that this chimeric dynein moves with biased small stepping motion in which only backward steps are slightly suppressed compared to forward and side steps.…”

De Novo Design of Allosteric Control into Rotary Motor V1-ATPase by Restoring Lost Function

“…In the 120° step rotation, the designed V1 was found to have the main-pauses and sub-pauses before the 40° and 80° sub-steps respectively, which is the same as the wild-type V1 28 (Fig. 5a).…”

“…The designed B-subunit (De), expressed with the A-subunit in E. coli using the plasmid pTR19-AB 28 and purified by a Ni 2+ -affinity chromatography followed by size exclusion chromatography, formed a ring complex with the A-subunit (A3(De)3 ring complex) ( Supplementary Fig. 7a).…”

“…From the results described above, a rotation scheme for the designed V1 is proposed based on the scheme for the wild-type recently proposed by Iida et al (Fig. 5e, left) 28 . For the wild-type, two or three catalytic sites are occupied at any time with ATP or its product(s) of hydrolysis.…”

Correlation between the numbers of rotation steps in the ATPase and proton-conducting domains of F- and V-ATPases

“…To date, there are only two wellcharacterized V-ATPases for which the number of rotational steps in V 1 and number of proton-carrying units in V o is known. One of them is the Enterococcus hirae V-ATPase (EhV), with a 6-step V 1 (Iida et al 2019) and a V o with 10 proton-carrying units (Murata et al 2005), providing support for the abovementioned correlation. The other one is the V-ATPase from Thermus thermophilus (ThV), which consists of a 3-step V 1 (Furuike et al 2011) and a V o with 12 proton-carrying units (Toei et al 2007).…”