Background:The chemomechanical coupling scheme of the rotary motor V 1 -ATPase is incompletely understood. Results: Enterococcus hirae V 1 -ATPase (EhV 1 ) showed 120°steps of rotation without substeps, as commonly seen with F 1 -ATPase. Conclusion:The basic properties of rotary dynamics of EhV 1 are similar to those of Thermus thermophilus V 1 -ATPase. Significance: This study revealed the common properties of V 1 -ATPases as rotary molecular motors, distinct from those of F 1 -ATPases.

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (k), translational movement (k), and dissociation (k) with single-molecule fluorescence imaging. The k for a single chitin microfibril was 2.1 × 10 M μm s. The k showed two components, k (3.2 s, 78%) and k (0.38 s, 22%), corresponding to bindings to different crystal surfaces. From the k, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10 M μm and 8.1 × 10 M μm, respectively. The k was 52.5 nm s, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s) calculated from k and biochemically determined low k (2.6 s) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s × 0.048 = 2.5 s). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

F 1 -and V 1 -ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F 1 or of V 1 and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F 1 or V 1 . These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F 1 and V 1 . The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F 1 -FliJ chimera generates only 10% of WT F 1 , the V 1 -FliJ chimera generates torque comparable to that of V 1 with the native axle protein that is structurally more similar to FliJ than the native rotor of F 1 . This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F 1 .rotary molecular motor | protein design | ATPase | F 1 | V-ATPase

V 1 -ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A 3 B 3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the highresolution molecular structure of Enterococcus hirae V 1 -ATPase (EhV 1 ) and revealed how the three catalytic sites in the stator A 3 B 3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV 1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV 1 and discuss a possible model of its chemomechanical coupling scheme. V C 2014 IUBMB Life, 66(9): [624][625][626][627][628][629][630] 2014