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

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.

Metastable asymmetrical structure of a shaftless V1 motor.

V1-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A3B3 complex and a central DF shaft. The nucleotide-free A3B3 complex of Enterococcus hirae, composed of three identical A1B1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A3B3. The nucleotide-free mutant A3B3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A3B3 forms a metastable (stressed) asymmetric structure composed of unstable A1B1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

Off-axis rotor in Enterococcus hirae V-ATPase visualized by Zernike phase plate single-particle cryo-electron microscopy.

EhV-ATPase is an ATP-driven Na+ pump in the eubacteria Enterococcus hirae (Eh). Here, we present the first entire structure of detergent-solubilized EhV-ATPase by single-particle cryo-electron microscopy (cryo-EM) using Zernike phase plate. The cryo-EM map dominantly showed one of three catalytic conformations in this rotary enzyme. To further stabilize the originally heterogeneous structure caused by the ATP hydrolysis states of the V1-ATPases, a peptide epitope tag system was adopted, in which the inserted peptide epitope sequence interfered with rotation of the central rotor by binding the Fab. As a result, the map unexpectedly showed another catalytic conformation of EhV-ATPase. Interestingly, these two conformations identified with and without Fab conversely coincided with those of the minor state 2 and the major state 1 of Thermus thermophilus V/A-ATPase, respectively. The most prominent feature in EhV-ATPase was the off-axis rotor, where the cytoplasmic V1 domain was connected to the transmembrane Vo domain through the off-axis central rotor. Furthermore, compared to the structure of ATP synthases, the larger size of the interface between the transmembrane a-subunit and c-ring of EhV-ATPase would be more advantageous for active ion pumping.

Head-to-Head Prenyl Synthases in Pathogenic Bacteria.

Many organisms contain head-to-head isoprenoid synthases; we investigated three such types of enzymes from the pathogens Neisseria meningitidis, Neisseria gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene, and we solved an inhibitor-bound structure that revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the first half of the reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis, we concluded that the Neisseria proteins were HpnDs (PSPP synthases). The differences in chemical reactivity to CrtM were due, at least in part, to the presence of a PSPP-stabilizing arginine in the HpnDs, decreasing the rate of dehydrosqualene biosynthesis. These results show that not only S. aureus but also other bacterial pathogens contain head-to-head prenyl synthases, although their biological functions remain to be elucidated.

Chemomechanical Coupling in Hexameric Protein-Protein Interfaces Harnesses Energy within V-Type ATPases.

ATP synthase is the most prominent bioenergetic macromolecular motor in all life forms, utilizing the proton gradient across the cell membrane to fuel the synthesis of ATP. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, the precise molecular mechanism whereby vacuolar (V-type) ATP synthase fulfills its biological function remains largely fragmentary. Recently, crystallographers provided the first high-resolution view of ATP activity in Enterococcus hirae V1-ATPase. Employing a combination of transition-path sampling and high-performance free-energy methods, the sequence of conformational transitions involved in a functional cycle accompanying ATP hydrolysis has been investigated in unprecedented detail over an aggregate simulation time of 65 µs. Our simulated pathways reveal that the chemical energy produced by ATP hydrolysis is harnessed via the concerted motion of the protein-protein interfaces in the V1-ring, and is nearly entirely consumed in the rotation of the central stalk. Surprisingly, in an ATPase devoid of a central stalk, the interfaces of this ring are perfectly designed for inducing ATP hydrolysis. However, in a complete V1-ATPase, the mechanical property of the central stalk is a key determinant of the rate of ATP turnover. The simulations further unveil a sequence of events, whereby unbinding of the hydrolysis product (ADP + Pi) is followed by ATP uptake, which, in turn, leads to the torque generation step and rotation of the center stalk. Molecular trajectories also bring to light multiple intermediates, two of which have been isolated in independent crystallography experiments.