Abstract
V-ATPases are rotary motor proteins which convert chemical energy of ATP into electrochemical potential of ions across the cell membrane. V-ATPases consist of two rotary motors, Vo and V1, and Enterococcus hirae V-ATPase (EhVoV1) actively transports Na+ in Vo (EhVo) by using torque generated by ATP hydrolysis in V1 (EhV1). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhVoV1 wild-type, aE634A, and BR350K mutants under the various Na+ and ATP concentrations ([Na+] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na+] was low and [ATP] was high, under the condition that only Na+ binding to EhVo is the rate-limiting, wild-type and aE634A exhibited 10-pausing positions reflecting 10-fold symmetry of the EhVo rotor and almost no backward steps. Duration time before forward steps was inversely proportional to [Na+], confirming that Na+ binding triggers the steps. When both [ATP] and [Na+] were low, under the condition that both Na+ and ATP bindings are rate-limiting, aE634A exhibited 13-pausing positions reflecting 10- and 3-fold symmetries of EhVo and EhV1, respectively. Distribution of duration time before forward step was well fitted by a sum of two exponential decay functions with distinct time constants. Furthermore, frequent backward steps smaller than 36° were observed. Small backward steps were also observed during long, three ATP cleavage pauses of BR350K. These results indicate that EhVo and EhV1 do not share pausing positions and Na+ and ATP bindings occur at different angles, and the coupling between EhVo and EhV1 is not elastic but rigid.
Significance Statement V-ATPases are ion pumps consisting of two rotary motor proteins Vo and V1, and actively transport ions across the cell membrane by using chemical energy of ATP. To understand how V-ATPases transduce the energy in the presence of structural symmetry mismatch between Vo and V1, we simultaneously visualized rotational pauses and forward and backward steps of Vo and V1 coupled with ion transport and ATP hydrolysis reaction, respectively. Our results indicate rigid coupling of a V-ATPase which has multiple peripheral stalks, in contrast to elastic coupling of F-ATPases with only one peripheral stalk, which work as ATP synthase. Our high-speed/high-precision single-molecule imaging of rotary ATPases in action will pave the way for a comprehensive understanding of their energy transduction mechanisms.
Introduction
Rotary ATPases are ubiquitously expressed in living organisms and play important roles in biological energy conversions (1-6). These rotary ATPases are classified into F-, V-, and A-ATPases based on their amino acid sequences and physiological functions (6). Eukaryotic and bacterial F-ATPases (FoF1), and archaeal A-ATPases (AoA1) mainly function as ATP synthases driven by electrochemical potential of ion across the cell membrane, although they can also act as active ion pumps driven by ATP hydrolysis depending on the cellular environments. On the other hand, V-ATPases (VoV1) in eukaryotes primarily function as active ion pumps. V-ATPases are also found in bacteria, and some of them are termed as V/A-ATPases based on their physiological function, ATP synthesis (6, 7).
To date, numerous studies have been conducted to understand how the two motor proteins (i.e., F1/A1/V1 and Fo/Ao/Vo) of the rotary ATPases couple their rotational motions and functions. Single-molecule studies using fluorescent probes (8-11), gold nanoparticle (AuNP) or nanorod probes (12-20), and Förster resonance energy transfer (FRET) (15, 21, 22) have revealed rotational dynamics of the rotary ATPases for both ATP hydrolysis/synthesis directions. Furthermore, recent cryo-electron microscopic (cryo-EM) single-particle analyses revealed entire architectures of the rotary ATPases with different structural states at atomic resolutions (23-34). In particular, several studies have demonstrated elastic coupling of FoF1 due to large deformations of the peripheral stalk connecting Fo and F1 (24, 28, 34). However, few studies on other types of rotary ATPases with different functions and subunit compositions have been performed, and a comprehensive understanding of the energy transduction mechanism remains elusive.
Enterococcus hirae V-ATPase (EhVoV1) works as an ATP-driven sodium ion (Na+) pump to maintain Na+ concentration ([Na+]) inside the cell (Figure 1A) (35-39). Note that we use the term V-ATPase or VoV1 because its physiological function is not ATP synthesis but active ion transport. EhVoV1 is a multi-subunit complex composed of 9 different subunits, namely, ac10dE2G2 and A3B3DF complexes in EhVo and EhV1, respectively. In EhV1 A3B3DF complex, three pairs of the A and B subunits form a hetero-hexameric A3B3 stator ring, and the central rotor DF subcomplex is inserted into the A3B3 ring (Figure 1B, bottom) (40, 41). The EhVo ac10dE2G2 complex transports Na+ across the cell membrane. The membrane-embedded rotor ring is formed by a decamer of tetra-helical transmembrane c-subunit (c10-ring, Figure 1B, top), connected with the central DF stalk via the d-subunit (25, 42). The stator a-subunit works as an ion channel, and two EG peripheral stalks interact with the a-subunit and A3B3 ring to assure rotary coupling between EhVo and EhV1. In EhV1, the ATP hydrolysis reaction is catalyzed at the interfaces of three A and B subunits. It drives a counterclockwise rotation of the DF rotor subunits as viewed from EhVo side (Figure 1B, bottom). Likewise other F1/A1/V1 (10, 43), EhV1 is a stepping motor that rotates 120° per one ATP hydrolysis (44, 45). By using high-speed and high-precision single-molecule imaging analysis with AuNP as a low-load probe, we previously revealed that the 120° step of isolated EhV1 is further divided into 40 and 80° substeps, which are triggered by ATP binding and ADP release, respectively (44). In contrast, as our previous single-molecule observation of EhVoV1 did not resolve clear pauses and steps (16), elementary steps in the rotation of EhVoV1 have not been revealed.
Although the mechanism of ion transport in the Fo/Ao/Vo has not been fully understood, the so-called “two-channel” model has been widely accepted (46-52). In this model, the a-subunit has two half-channels for ion entry/exit into/from the ion-binding sites of rotor c-ring. In the case of EhVo, Na+ enters the half-channel from the cytoplasmic side and binds to the negatively charged Na+-binding sites of the c-subunit (42, 53). Then, the charge-neutralized c-subunit can move into the hydrophobic lipid membrane (51, 54). The rotational torque generated by ATP hydrolysis in EhV1 is transmitted to EhVo via the rotor d-subunit, allowing the c10-ring to rotate unidirectionally in the lipid membrane. Na+ translocated by a nearly single turn of the c10-ring reaches another half-channel of the a-subunit, which connects the Na+-binding site of c-subunit to the extracellular side. Then, Na+ is pumped out of the cell by a hydrated microenvironment (55) and/or the electrostatic repulsion with the positively charged residues in the a-subunit, aArg573 and aArg629, located at the interface between two half-channels (Figures 1C and S1) (25). Because EhVoV1 has the c10-ring, ten Na+ are transported per single turn. Therefore, the step size of EhVo is expected to be 36° (360°/10), similar to Escherichia coli (E. coli) and yeast FoF1, which also have c10-rings (12, 13, 21, 34).
The ion-to-ATP ratio is a central issue in the coupling mechanism of rotary ATPases. All known F1/A1/V1 has three catalytic sites and 3-fold structural symmetry, and hydrolyzes or synthesizes 3 ATP molecules per single turn. In contrast, the number of protomers forming the rotor c-ring of Fo/Ao/Vo varies from 8 to 17 depending on the species, suggesting wide variations in the ion-to-ATP ratio of rotary ATPases (56, 57). In EhVoV1, because the rotor c-ring of EhVo has a 10-fold structural symmetry (Figure 1B, top), this enzyme has a structural symmetry mismatch and a non-integer ratio between transported Na+ and hydrolyzed ATP (10/3 = 3.3). If the rotational coupling between EhVo and EhV1 is elastic, as reported for E. coli and yeast FoF1, the symmetry mismatch is relieved by large deformations of the peripheral stalk and/or the central rotor (24, 28, 34, 58). On the other hand, if the coupling is rigid due to multiple peripheral stalks of the EhVoV1, the pausing positions of both EhVo and EhV1 would be observed independently in a single-molecule observation. To address this issue, it is required to directly visualize the rotational pauses and steps of EhVoV1 under the conditions that elementary steps of rotation for EhVo and EhV1 are both rate-limiting, for example, the bindings of Na+ and ATP to EhVo and EhV1, respectively.
Here we carried out high-speed and high-precision single-molecule imaging of rotation of detergent-solubilized EhVoV1 by using 40-nm AuNP as a low-load probe. To resolve the rotational pauses and steps of EhVo, a glutamate residue in the stator a-subunit (aGlu634) was replaced with alanine. Since the mutated aGlu634 is located on the surface of Na+ entry half-channel (Figures 1C and S2), Na+ binding to the c-subunit in EhVoV1(aE634A) mutant (hereinafter, referred to as aE634A) is expected to become slower than in wild-type. The rotation rate of aE634A decreased about 10 times compared with that of the wild-type, allowing us to clearly resolve the rotational pauses and steps in EhVo. Under the condition that only Na+ binding is the rate-limiting, aE634A showed 10 pausing positions per single turn and a step size of about 36°, consistent with 10 protomers in the c10-ring of EhVo. The duration time before the forward step was inversely proportional to the [Na+], indicating that the dwell corresponds to the waiting time for Na+ binding. On the other hand, under the condition that both Na+ and ATP bindings are the rate-limiting, 13 pausing positions per single turn were observed. Furthermore, backward steps smaller than 36° were frequently observed only when ATP binding is also the rate-limiting, indicating that EhVoV1 undergoes Brownian motion between adjacent pausing positions of EhVo and EhV1 when no torque is applied from EhV1. The backward steps of 36° or larger than 36° were rarely observed, suggesting the suppression of the reverse Na+ transport. Small backward steps were also frequently observed during long, three ATP cleavage pauses of another mutant EhVoV1(BR350K) in which ATP hydrolysis is the rate-limiting of rotation (44). From these results, we conclude that EhVo and EhV1 do not share their pausing positions and Na+ and ATP bindings occur at different angles, and their coupling is not elastic but rigid.
Results
Single-molecule imaging system and generation of EhVoV1 aE634A mutant
For the single-molecule observation, a poly-histidine tag (His3-tag) was introduced to the C-terminus of c-subunit to immobilize the detergent-solubilized EhVoV1 complex on a Ni2+-NTA coated glass surface (Figure 2A). Furthermore, the N-terminus of A-subunit was biotinylated by a genetically fused Avi-tag (59), and streptavidin-coated AuNP probe (φ=40 nm) was attached (Figure 2A). Note that the stator subunits rotate against the rotor subunits by ATP hydrolysis in our experimental system because the rotor c10-ring was fixed on the glass surface. The ATP-driven rotation was observed under the total internal reflection dark-field microscope at 1,000 or 3,000 frames per second (fps) at 25°C. In this system, the localization precision was 0.6 nm at 3,000 fps (0.33 ms temporal resolution), determined by centroid analysis of the scattering images of single AuNPs non-specifically attached to the glass surface (Figure S3).
To clearly visualize the ATP-driven rotation rate-limited by Na+ transport, site-directed mutagenesis was conducted on the a-subunit of EhVo. In the a-subunit of Vo, there are some negatively charged amino acid residues on the surface of half-channels forming ion transport pathways (23, 60). In EhVo, aGlu634 is located on the Na+ entry half-channel (Figure 1C). This glutamate residue is completely conserved among other VoV1 (Figure S1), suggesting that this negatively charged residue has a role in attracting ions into the entry half-channel. To reduce the Na+ binding rate, we prepared EhVoV1 mutant, aE634A, where aGlu634 was replaced with alanine to eliminate the negative charge of the side chain. The purified aE634A solubilized with n-dodecyl-β-D-maltoside (DDM) exhibited the subunit stoichiometry similar to the wild-type in SDS-PAGE (Figure S4), indicating intactness of the complex.
[ATP] and [Na+] dependence of rotation rate of wild-type and aE634A
Rotation rates of wild-type and aE634A at various substrate concentrations were examined from the slope of the time course of rotation (Figures 2B, C, and S5). [ATP] dependences of the rotation rate for wild-type and aE634A in the presence of 300 mM Na+ are shown in Figure 2B. Under this high [Na+] condition, Na+ binding is fast and not rate-limiting, and consequently, [ATP] dependence obeyed Michaelis-Menten kinetics. Obtained kinetic parameters, the Michaelis constant (KmATP) and the maximum velocity (VmaxATP), are summarized in Table 1. The value of KmATP for wild-type was 60.4 ± 2.1 μM (fitted value ± S.E. of the fit). This value was comparable to that for isolated EhV1 (43 ± 6 μM), indicating that apparent affinity of ATP was not significantly affected by EhVo. On the other hand, the value of VmaxATP (40.0 ± 0.3 rps, fitted value ± S.E. of the fit) was significantly smaller than that of the isolated EhV1 (117 ± 3 rps) (44). As a result, the binding rate constant of ATP (konATP) estimated by 3 × VmaxATP / KmATP was smaller than that of the isolated EhV1 (Table 1). We attributed this difference to the intact interaction between rotor c10-ring and stator a-subunit of EhVo and concluded that Na+ transport in EhVo limits the VmaxATP of EhVoV1, as discussed in our previous single-molecule study (16). In aE634A, the values of KmATP and VmaxATP were 6.6 ± 0.3 μM and 4.58 ± 0.04 rps, respectively, and both were approximately one-tenth of those for wild-type (Table 1). Thus, the konATP for aE634A (2.1 × 106 M-1s-1) was in close agreement with that for wild-type (2.0 × 106 M-1s-1) (Table 1). These results indicate that the mutation in aGlu634 affects only Na+ transport but not ATP binding in the EhVoV1 complex.
Figure 2C shows [Na+] dependence of the rotation rate at the saturated [ATP] (5 mM). Here, contaminated Na+ in the observation buffers were estimated by an inductivity coupled plasma optical emission spectrometer (ICP-OES) (Figure S6), and total [Na+] including contamination was used for the plot. Under this condition, [ATP] is sufficiently high and ATP binding is not rate-limiting (Figure 2B and Table 1), and the effect of [Na+] on the rotation rate becomes obvious. As a result, both wild-type and aE634A showed biphasic responses to [Na+]. As reported in our previous studies, data were fitted by a summation of two independent Michaelis-Menten equations assuming two Na+ binding sites (16, 61). The obtained kinetic parameters are shown in Table 2. The both values of Vmax1Na and Vmax2Na for aE634A (1.1 ± 0.3 and 4.4 ± 0.5 rps, fitted value ± S.E. of the fit) were about ten times smaller than those for wild-type (15.7 ± 1.6 and 35.0 ± 7.1 rps) (Table 2). On the other hand, considering the large fitting error especially in wild-type, the values of Km1Na and Km2Na do not seem to be significantly different between aE634A (0.32 ± 0.28 and 58.3 ± 26.5 mM, fitted value ± S.E. of the fit) and wild-type (0.29 ± 0.09 and 160.2 ± 88.8 mM). Then, the binding rate constant of Na+ (konNa) estimated by 10 × VmaxNa / KmNa largely decreased in aE634A, especially more than ten times for the high-affinity site (kon1Na, Table 2). The significant decrease in the Vmax2Na for aE634A compared with that of wild-type also suggests that the rate of elementary reaction steps other than Na+ binding also decreased in EhVo. Because Na+ binding to EhVo is certainly the rate-limiting at low [Na+] for both wild-type and aE634A, we then conducted a detailed analysis of the rotational pauses and steps at low [Na+].
Pauses and steps of EhVo and EhV1 in ATP-driven rotation of wild-type and aE634A
Figures 3A and S7 show typical time courses of rotational trajectory (pink line) of wild-type observed at 0.3 mM Na+ and 5 mM ATP with 3,000 fps (0.33 ms temporal resolution). Enlarged trajectory for single revolution and corresponding x-y trajectory (inset, pink line) are also shown. Under this condition, not ATP binding but Na+ binding is the rate-limiting of ATP-driven rotation (Figures 2B and C, Table 2). The rotational trajectory showed transient pauses and steps in a forward, counterclockwise direction. Then, the pauses and steps were objectively detected with a step-finding algorithm (62) (black line on the trajectory) applied to median-filtered traces (current ± 4 frames, red line), as our previous studies on other motor proteins (63, 64). As indicated by the numbers in the trajectories, 10 pauses were mainly detected in a single turn (Figure S8A), reflecting the number of protomers in rotor c10-ring (Figure 1B, top). In addition, number of pauses smaller or larger than 10 per single turn was also detected (Figure S8A). The number of pauses smaller than 10 can be attributed to undetectable short pauses due to the limited time resolution, because stepping behavior is a stochastic process. The number of pauses larger than 10 is presumably attributed to occasional detection of the sub-pauses of EhV1 for ADP release, which has a time constant of 2.5 ms and is independent of [ATP] (44). Furthermore, small fraction of under-or over-fitting of the step-finding algorithm would also be included (64). Distribution of the step size revealed that backward steps were rarely observed (0.8%, Figure 3B), the average value of forward step was 36.6°, and a Gaussian function well fitted the distribution with the peak at 33.8 ± 14.2° (peak ± S.D.). These values show excellent agreement with the step size expected from the structural symmetry of c10-ring (360°/10=36°). Distribution of the duration time before forward step was well fitted by a single exponential decay function with the time constant of 13.5 ± 0.3 ms (fitted value ± S.E. of the fit), indicating single rate-limiting step of the rotation (Figure 3C).
Rotational trajectories of aE634A observed at 0.3 mM Na+ and 5 mM ATP with 1,000 fps are shown in Figures 4A and S9. In aE634A, the median filtering of current ± 7 frames was applied for the step-finding algorithm (62). As in wild-type, the number of pauses per single turn was mainly 10 (Figure S8B), and the step size was 37.1° (average) or 34.9 ± 12.8° (peak ± S.D.) (Figure 4B), consistent with the structural symmetry of c10-ring, and the ratio of backward step was minor (1.2%). Distribution of the duration time before forward steps for aE634A was well fitted by a single exponential decay function with the time constant of 137 ± 6 ms (Figure 4C) which is 10 times larger than that for wild-type, consistent with the analysis of rotation rate (Figure 2C and Table 2). For aE634A, we also examined [Na+] dependence, and Figure S10 shows rotational trajectories at 0.09 or 1.3 mM Na+ and 5 mM ATP. Distributions of the step size and the duration time before forward step at 0.09 and 1.3 mM Na+ are shown in Figure 5A-D, respectively. As same at 0.3 mM Na+, the step sizes of about 36° were obtained, indicating that 36° step is independent of [Na+]. In contrast, although distributions of the duration time before forward step were also well fitted by single exponential decay functions as 0.3 mM Na+, the time constants changed largely. Figure 5E shows [Na+] dependence of the time constant. The slope was −1.1, in the case that the data point at 1.3 mM Na+ was excluded because it is the first saturating concentration observed in the rotation rate analysis (Figure 2C and Table 2). If this data point was included, the slope was −1.0. Therefore, the time constant is almost inversely proportional to [Na+], indicating that Na+ binding is the rate-limiting of rotation. Furthermore, this result indicates that ATP hydrolysis in EhV1 is tightly coupled with Na+ transport in EhVo in the EhVoV1 complex solubilized with DDM.
Next, we observed rotation of aE634A at 0.3 mM Na+ and 1 μM ATP with 1,000 fps (Figures 6 and S11). Under this condition, both Na+ and ATP bindings are the rate-limiting of rotation (Figures 2B and C). The step-finding algorithm was applied to the median-filtered trace (current ± 7 frames, red line). As a result, the number of detected pauses in a single turn was mainly 13, reflecting 10-pausing positions of EhVo and 3-pausing positions of EhV1 (Figure S8C). This result indicates that the pausing positions waiting for Na+ and ATP bindings are different in EhVoV1 complex. Distribution of the step size is shown in Figure 6B. The average value for forward step was 29.9° and distinctly smaller than 36°. When we superimposed two histograms of Figure 4B (0.3 mM Na+ and 5 mM ATP) and Figure 6B (0.3 mM Na+ and 1 μM ATP) after normalizing the maximum values, difference was obvious (Figure S12). Furthermore, interestingly, in contrast to the condition that Na+ binding was the sole rate-limiting factor, the ratio of backward step increased to 6.1%. Therefore, we fitted the distribution with the sum of three Gaussians: one peak in backward (minus) direction and two peaks in forward (plus) direction. Note that one of the forward peaks was fixed at 36°, assuming as the step of EhVo. Then, we obtained three peaks at −14.2 ± 6.8, 23.1 ± 10.4, and 36.0 ± 12.8° (peak ± S.D., Figure 6B). For comparison, the fitting with two Gaussians with peaks at minus and plus directions is shown in Figure S13A. Distribution of the duration time before forward step was better fitted with a sum of two exponential decay functions (coefficient of determination R2 = 0.98, Figure 6C) than with a single exponential decay function (R2 = 0.94, Figure S13B), and time constants of 135 ± 24 and 642 ± 322 ms (fitted value ± S.E. of the fit) were obtained. These values presumably correspond to the time constants for Na+ and ATP bindings, respectively, because the time constant for Na+ binding was 137 ms at 0.3 mM Na+ and 5 mM ATP (Figure 4C), and that for ATP binding was 476 ms at 1 μM ATP estimated from konATP (2.1 × 106 M-1s-1, Table 1). These results are consistent with the notion that both Na+ and ATP bindings are the rate-limiting of rotation.
Backward and recovery steps between adjacent pauses of EhVo and EhV1
As described above, frequent backward steps (6.1% of total steps) were observed under the condition that both Na+ and ATP bindings are the rate-limiting of rotation (Figure 6B). Typical examples of the backward step are shown in Figure 7. Green, cyan, and purple lines indicate forward step just before the backward step, backward step, and forward step after the backward step (recovery step), respectively. Figures 7B-D show the step size distributions for these events. The average value of backward step size was −18.8° (Figure 7C), which is smaller than the expected step size (36°) of EhVo. Because backward steps were frequently observed when not only Na+ binding but also ATP binding was the rate-limiting of rotation, it is reasonable to assume that backward steps occur during the pauses waiting for ATP binding. The backward step size smaller than 36° is also consistent with the notion that the pausing positions waiting for Na+ and ATP bindings are different in EhVoV1 complex, and backward steps occur at the intermediate pausing position of EhV1 between pausing positions of EhVo. The forward steps just before the backward and recovery steps seemed to show two kinds of step sizes, smaller than 36° and close to 36° (Figures 7B and D). The backward and forward steps smaller than 36° suggest the Brownian motion between the adjacent pausing positions waiting for ATP and Na+ bindings. Furthermore, 36° forward steps just before the backward steps suggest a standard forward step in EhVo followed by the Brownian backward step to the adjacent pausing position of the EhV1, and 36° recovery steps after the backward steps suggest a resumption of the rotation after ATP binding to EhV1.
Then, to test the hypothesis whether the backward steps occur equally at three pausing positions of EhV1, we prepared other mutants, isolated EhV1(BR350K) and EhVoV1(BR350K) complex. Because an arginine residue in the B-subunit (BArg350) of EhV1 plays a crucial role in ATP hydrolysis (Figure S14) (40), the rotation rate of EhV1(BR350K) mutant decreases about 100 times than that of wild-type EhV1 (44). This property makes ATP cleavage pauses of EhV1 much longer in EhVoV1(BR350K) even at high [ATP], and the pauses of EhVo and EhV1 become distinguishable. The rotations of EhV1(BR350K) and EhVoV1(BR350K) observed at 0.3 mM Na+ and 5 mM ATP with 1,000 fps are shown in Figure S15. Note that in the isolated EhV1(BR350K), His6-tag was introduced to the N-terminus of the A-subunit to immobilize the stator A3B3 ring, and 40-nm AuNP probe was attached to the rotor D-subunit (Figure S14). Three long pauses were observed in both mutants, attributed to ATP cleavage pauses. Similar to our previous study, EhV1(BR350K) did not show clear backward steps during long ATP cleavage pauses under this condition (Figure S15A) (44). On the other hand, EhVoV1(BR350K) exhibited frequent backward and recovery steps during long ATP cleavage pauses (Figure S15B). Distribution of the step size showed two peaks at −11 and 11° and the sizes were smaller than 36° (Figure S15C), and these small backward and recovery steps occurred equally in all three ATP cleavage pauses of EhV1.
Discussion
ATP-driven rotation of EhVoV1 tightly coupled with [Na+]
In the present study, we directly visualized ATP-driven rotation of EhVoV1 rate-limited by ion transport. EhVoV1 used in this study transports Na+, while most of the rotary ATPases transport H+. This property of EhVoV1 has advantages to resolve the stepping rotation rate-limited by ion transport in Vo. Firstly, H+ transport in rotary ATPases is thought to be achieved by a Grotthuss mechanism (14, 65), in which H+ transfer is not a rate-limiting process. Indeed, the duration time of transient dwells of Fo in E. coli FoF1 did not exhibit pH dependence ranging from pH 5 to 9, although the frequency of pause occurrence was highly affected by pH (14). In addition, changes in pH can largely affect protein stabilities and enzymatic reactions, whereas changes in [Na+] would mildly affect them. Here, we resolved the pauses and steps of EhVoV1 by using a mutant in which aGlu634 residue in the stator a-subunit of EhVo was substituted with alanine (aE634A) and found that the duration time before forward steps is inversely proportional to [Na+] (Figure 5E). We have previously confirmed that the coupling in EhVoV1 is retained by using DCCD (N, N’-dicyclohexylcarbodiimide) modification assay (16). [Na+] dependences of the rotation rate in both wild-type and aE634A (Figure 2C) are also strong evidence for the intact coupling. To the best of our knowledge, the present study first demonstrated ATP-driven rotation of a rotary ATPase rate-limited by the ion transport and tight coupling between ion binding and rotational step, although single-molecule studies of ATP-driven rotation of H+-transporting rotary ATPases have been reported (12-14, 66-69).
We found that both wild-type and aE634A mainly shows 10 pauses per single turn and 36° steps (Figures 3 and 4), which reflect the structural symmetry of c10-ring under the condition that only Na+-binding is the rate-limiting of rotation (0.3 mM Na+ and 5 mM ATP). In addition, number of pauses larger or smaller than 10 per single turn was also observed (Figure S8). From the time constants for elementary steps of ATP hydrolysis reaction in EhV1 and the time resolution of measurement (0.33 and 1 ms for wild-type and aE634A, respectively), we can consider the case where more than 10 pauses are detected (Figure S8). From the konATP of wild-type and aE634A (2.0×106 and 2.1×106 M-1s-1, respectively, Table 1), the time constants for ATP binding are estimated to be about 0.1 ms at 5 mM ATP. In addition, by using the isolated EhV1, we previously reported that the time constants for ATP cleavage and phosphate release are smaller than 1 ms, and that for ADP release is 2.5 ms. Furthermore, ADP release occurs at different angle from that of ATP binding, while ATP cleavage and phosphate release occur at same angle to ATP binding (44). Considering stochastic nature of pause duration and current time resolution, these elementary events, especially ADP release, can be also detected. If the time resolution and the localization precision is further improved, detection of 16 pauses (10, 3, and 3 pauses for Na+ binding, ATP binding, and ADP release, respectively) would become possible, and give us further insight into the energy transduction mechanism of EhVoV1 complex. Similarly, number of pauses less than 10 can also occur stochastically due to Na+ binding events with shorter dwell time than the time resolution. Another possible explanation is stochastic H+ transport instead of Na+ in EhVoV1 at low [Na+]. It has been reported that H+ can bind to the ion-binding site of EhVo c-subunit when [Na+] is sufficiently low (42). Because H+ binding will not be the rate-limiting factor as described above, the pauses could not be detected at the current time resolution (13). In the present study, we could not distinguish Na+ and H+ bindings, and assumed that steps are triggered by Na+ bindings. Whether or not EhVoV1 really transports H+ under the low [Na+] conditions will be discussed elsewhere. Furthermore, we need to mention that the step-finding algorithm we used cannot perfectly eliminate the over-and under-fittings (62, 64).
The VmaxATP of wild-type EhVoV1 (40.0 ± 0.3 rps) at 300 mM Na+ was smaller than that of isolated EhV1 (117 ± 3 rps) (Figure 2B and Table 1) (44). The difference is presumably caused by elementary steps of Na+ transport in EhVo, especially by Na+ release, and/or the interaction (molecular friction) between rotor c10-ring and stator a-subunit. If we assume that Na+ release is the rate-limiting, we can estimate the time constant for Na+ release to be 1.6 ms from the difference of the VmaxATP between EhVoV1 and EhV1. Note that in the present study, both entry and exit half-channels of the a-subunit were exposed to the solution with 300 mM Na+ because we used detergent-solubilized EhVoV1. Effect of Na+ rebinding from the exit half-channel on the rotation rate of EhVoV1 is an interesting question that needs to be clarified in future. On the other hand, the VmaxATP of aE634A (4.58 ± 0.04 rps) at 300 mM Na+ was approximately one-tenth that of wild-type (40.0 ± 0.3 rps) (Figure 2B and Table 1). Michaelis-Menten kinetics analysis showed that the konATP of aE634A was similar to that of wild-type, although the konNa of aE634A significantly decreased compared with that of wild-type (Tables 1 and 2). These results indicate that aE634A mutation has little effect on ATP binding to EhV1. The mutated glutamate residue is located on the surface of entry half-channel of a-subunit (Figure 1C), and the decrease in konNa is likely caused by the loss of negative charge. Interestingly, this glutamate residue is completely conserved among Na+-and H+-transporting V-ATPases (Figures S1 and S2) (23, 25, 60, 70). Therefore, it may have a common role in efficient ion uptake for both Na+ and H+. Consistent with this notion, ion selectivity seems to be determined by the ion-binding site of c-ring rather than the half-channels in a-subunit (71, 72).
The rotation rate of both wild-type and aE634A showed a biphasic response to [Na+] (Figure 2C and Table 2). This biphasic response was also reported previously on the ATPase activity of detergent-solubilized EhVoV1 (16, 61). The Michaelis-Menten parameters obtained in this study are slightly different from those in previous studies, presumably due to differences in experimental conditions, especially pH and [Na+] contaminated in the observation buffer. Regarding the Na+ contamination in the experimental system, it should be noted that it is difficult to discuss the affinity at the sub-micromolar range since the observation buffer contains at least 90 μM of Na+ (Figure S6). The biphasic response to [Na+] strongly suggests two different Na+-binding sites of EhVoV1 with largely different affinities. One possible explanation is that these sites are related to the entry and exit half-channels of a-subunit. The high-affinity binding site may correspond to the Na+ binding to the c-ring from the entry site because the dissociation constant of Na+ to c-subunit in EhVoV1 complex was reported as 12 μM in the absence of ATP (42). On the other hand, the low-affinity Na+ binding site corresponding to Km2Na has not been identified yet. We assume that the low-affinity site would be physiologically important because an intracellular [Na+] in Enterococcus hirae has been reported to be several tens of millimolar (36, 39, 73). To clarify the mechanism of the biphasic response of EhVoV1 to [Na+], it will be required to embed EhVoV1 in the lipid membrane and change the [Na+] in the entry and exit sides independently (74). Alternatively, mutagenesis in the Na+ exit half-channel of a-subunit will provide new insights into the biphasic response. Recently, Yanagisawa et al. investigated E. coli FoF1 mutants by replacing charged or polar residues in entry and exit half-channels with non-polar leucine residues and successfully revealed that these residues possess optimal pKa values for unidirectional H+ transfer (14). A similar approach would be also helpful for EhVoV1.
Rigid coupling between EhVo and EhV1 in the EhVoV1 complex
By using single-molecule imaging of aE634A under the condition that both Na+ and ATP bindings are the rate-limiting of rotation, we visualized the pauses of EhVo and EhV1 simultaneously (Figure 6A and S11). We found that aE634A mainly shows 13 pauses per single turn under this condition (Figure S8C). Furthermore, frequent backward and recovery steps smaller than 36° were observed (Figure 7), not only in aE634A but also during three long ATP cleavage pauses of EhVoV1(BR350K) (Figure S15). These results indicate that pausing positions waiting for Na+ and ATP bindings are different, and transitions occur between adjacent pausing positions of EhVo and EhV1. These results also indicate that ATP hydrolysis reaction in EhV1 is tightly coupled with Na+ transport in EhVo, and this coupling is not elastic but rigid. Considering our observation system where the stator aA3B3E2G2 complex rotates against the rotor dc10DF complex immobilized on the glass surface, if the complex shows a fully elastic coupling, the pausing positions of EhVo and EhV1 would be superimposed by deformation of the peripheral E2G2 stalks or the central DF shaft. In this case, single turn (360° revolution) would be divided into two 108° (36°×3) and one 144° (36°×4) with extensive deformation. However, our results clearly showed 10 pauses per single turn (36° step) at high [ATP] and 13 pauses at low [ATP], indicating the rigid coupling in which 120° steps in EhV1 are retained in the presence of structural symmetry mismatch between EhVo and EhV1.
The different pausing positions between Vo and V1 have also been reported by a single-molecule study of detergent-solubilized H+-transporting Thermus thermophilus V/A-ATPase (here termed as TtVoV1), which acts as ATP synthase (19). During rotation in the ATP-hydrolysis direction, TtVoV1 showed 30° steps derived from c12-ring of TtVo and extra pauses between two adjacent pauses of TtVo. These extra pauses were attributed to catalytic dwells of TtV1. Furthermore, corresponding structures were also recently revealed by cryo-EM study of nanodisc-embedded TtVoV1 (23). Zhou et al. resolved structures of two substates in addition to three main states, which reflect three-fold structural symmetry of TtV1. Those substates, 1L and 1R, which differ in the relative position of TtVo and TtV1, exhibited slight twist of central shaft and deformation of peripheral stalks against state 1. Although it remains uncertain which pauses found in the single-molecule study correspond to those substates, it is intriguing that the angles of TtVo and TtV1 do not coincide despite that TtVo has c12-ring and no symmetry mismatch with TtV1.
The rigid coupling between EhVo and EhV1 is distinct from E. coli, Bacillus PS3, and yeast mitochondrial F-ATPases, in which cryo-EM studies have proposed the elastic coupling (24, 28, 34). These F-ATPases have c10-rings (33, 75) and same structural symmetry mismatches as EhVoV1. Structural analysis of E. coli or Bacillus PS3 FoF1 revealed that three rotational steps were classified into two 108° steps and one 144° step. This significant mismatch was tolerated by the flexible peripheral stalk rather than the central shaft (24, 28, 76). Actually, very recent cryo-EM study of yeast mitochondrial FoF1 revealed extensive deformation of the peripheral stalk during catalysis (34). In rotary ATPases, number of the peripheral stalks varies from one to three, depending on enzymes (6, 77, 78). E. coli, Bacillus PS3 and yeast FoF1 possess only one peripheral stalk (24, 28), whereas EhVoV1 has two peripheral stalks which can strongly connect two motor domains (Figure 1A) (25). As a result, EhVoV1 would rotate rigidly without large and elastic deformations. Consistent with this notion, a cryo-EM study of yeast VoV1 which has three peripheral stalks showed almost uniform three 120° steps despite having a c10-ring (26).
In addition to the number of peripheral stalks, the number of c-subunits in a rotor ring also varies widely from 8 to 17 among the species (56, 57). This makes the issue more complicated, because different number of c-subunits will change the step size and the ion-to-ATP ratio. Furthermore, number of transmembrane helices in a c-subunit monomer also differs among species. For example, although both EhVoV1 and E. coli FoF1 have c10-ring, each c-subunit consists of tetra-and double-transmembrane helices, respectively (24, 53). This results in largely different diameters of c10-ring, 8 and 5 nm for EhVoV1 and E. coli FoF1, respectively (25). Obviously, larger rings would require larger deformations to adjust relative angles between Vo/Fo and V1/F1 if the elastic coupling is assumed. Another interesting feature of EhVoV1 with large c-ring is “off-axis” rotation which may affect the coupling between EhVo and EhV1 (25). However, the off-axis rotation was not resolved in the present single-molecule experiments. To understand the common and diverse mechanisms of energy transduction in the rotary ATPases, a comprehensive study of various ATPases with rotor c-rings of different c-subunit numbers and sizes will be required.
Brownian ratchet rotation of EhVo
Figure 8 shows the schematic model of the stepping rotation of EhVoV1 at high [ATP] and low [Na+] (Figure 8A), and at low [ATP] and low [Na+] (Figure 8B). In the present study, backward steps were rarely observed when [ATP] is high (5 mM) in both wild-type and aE634A (Figures 3B, 4B, 5A, and 5B). In EhVoV1, the torque for ATP-driven rotation is generated by ATP binding and ADP release in EhV1 (44), and the value has been estimated to be 20 pNnm (16). When [ATP] is high, time constants for ATP binding and ADP release are both small, and the torque from EhV1 will be applied to EhVo almost constantly. Therefore, EhVoV1 will show unidirectional rotation without backward steps (Figure 8A). On the other hand, when [ATP] is low (1 μM), where the time constant for ATP binding is large (476 or 642 ms, Table 1 and Figure 6C), frequent backward steps were observed (Figure 7A). The size of the backward steps was much smaller than 36° (14.2 or 18.8°, Figures 6B and 7C). Because no torque is applied when EhVoV1 is waiting for ATP binding at the pausing position of EhV1 (Figure 8B, dark green), it can move to the adjacent backward (and also forward) pausing position of EhVo by the Brownian motion (Figure 8B, blue arrows). Backward and recovery steps smaller than 36° were more frequently observed in EhVoV1(BR350K), in which ATP cleavage pauses are prominently longer than wild-type (Figure S15). Importantly, backward steps occurred equally during three ATP cleavage pauses of EhV1, consistent with the notion that backward steps are driven by thermal fluctuation, not by elastic strain accumulation. These results support Brownian ratchet model of EhVo rotation in addition to the rigid coupling of EhVoV1. Note that the size of backward steps occurring at three pausing positions of EhV1 will not be uniform in the model shown in Figure 8B. However, it was difficult for us to resolve the difference under the current experimental set up.
In our previous study of the isolated EhV1, no backward steps are observed in ATP-driven rotation except under extreme experimental conditions (44). Therefore, backward and forward steps smaller than 36° observed in EhVoV1 are caused by EhVo, and are presumably coupled with the Na+ binding/release to/from c-subunit through the two half-channels of a-subunit. A recent single-molecule study observed similar small backward steps of 11° in E. coli FoF1 during ATP-driven rotation and successfully revealed that backward steps are related to H+ translocation between c-ring and half-channels of a-subunit (14). In the study of E. coli FoF1, small backward step was attributed to the fact that E. coli FoF1 functions as ATP synthase. In the case of EhVoV1, because backward steps of 36° or larger than 36° rarely occurred, backward flow of Na+ seems to be suppressed. The detailed mechanisms of the backward step may be different by ATP synthesis capacity, size of the c-ring, and ion species being transported. In the present study, however, the electrochemical potential of Na+ could not be applied because detergent-solubilized EhVoV1 was used. To address the question of whether the electrochemical potential of Na+ can drive rotation of EhVoV1 in opposite direction and ATP synthesis, our next targets are single-molecule imaging and biochemical assay of EhVoV1 embedded in the lipid membrane.
Materials and Methods
Sample preparation
The construction of the expression plasmid for wild-type EhVoV1 (pTR19-EhVoV1) was reported previously (16). For the plasmid construction of aE634A, EhVoV1(BR350K), and EhV1(BR350K), PCR-based site-directed mutagenesis was performed by KOD one PCR Master Mix (Toyobo) with pTR19-EhVoV1 as a template. For expression and purification of wild-type, aE634A, EhVoV1(BR350K), and EhV1(BR350K), the procedures described in our previous study were used with some modifications (16, 44). Details are described in the SI Appendix.
Single-molecule imaging
Gold nanoparticle (AuNP) with a diameter of 40 nm (EMGC40, BBI) was biotinylated with biotin-alkane-thiol (HS-C11-EG3, Surfmods) and coated by Streptavidin (PRO791, PROSPEC) as described in the previous reports (44, 79). We followed the setting of a total internal reflection dark-field microscope system based on a high-speed CMOS camera and an inverted microscope as described in our previous studies with some modifications (44). Details are described in the SI Appendix.
ICP-OES measurement
To estimate the concentrations of contaminated Na+ in the observation buffers, we measured emission spectra using an inductivity coupled plasma optical emission spectrometer (ICP-OES; 5100 ICP-OES, Agilent Technologies). The emission signal at λ = 589.592 nm was used for analysis. The flow rates of plasma and assist gas (Ar) were 14 and 1.2 L/min, respectively. The standard addition method was used to avoid physical and ionization interferences. Namely, sodium standard solution (FUJIFILM Wako) was added to the observation buffers in the range of 0.1 to 10 mg/L (4.3 to 430 μM) as a final concentration of Na+. The calibration curve was extrapolated and the absolute value of the X-intercept was taken as the concentration of contaminated Na+.
Acknowledgments
We thank Monique Honsa for the advice about data analysis, Kazuyoshi Murata, Chihong Song, and Raymond N. Burton-Smith for providing us the structural data and their fruitful discussion, Yayoi Kon for her grateful technical support, and all laboratory members for helpful discussion and technical advice. This work was supported by the Grant-in-Aid for Scientific Research on Innovative Areas “Molecular Engine” (JP19H05380 to H.U., JP18H05425 to T.M., and JP18H05424 to R.I.), the Grants-in-Aid for Scientific Research (JP21H02454 to R.I., and JP21K15060 and JP20J01316 to A.O.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by the NINS program for cross-disciplinary study (Grant Number 01312001 to A.O.). A part of this work was performed with the aid of Instrument Center of the Institute for Molecular Science.
Footnotes
Competing Interest Statement: The authors declare no competing interest.