1. Structural Biology and Molecular Biophysics
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Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance

  1. Jun-ichi Kishikawa
  2. Atsuko Nakanishi
  3. Aya Furuta
  4. Takayuki Kato
  5. Keiichi Namba
  6. Masatada Tamakoshi
  7. Kaoru Mitsuoka
  8. Ken Yokoyama  Is a corresponding author
  1. Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Japan
  2. Institute for Protein Research, Osaka University, Japan
  3. Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Research Center for Ultra-High Voltage Electron Microscopy, Mihogaoka, Japan
  4. Graduate School of Frontier Biosciences, Osaka University, Japan
  5. RIKEN Center for Biosystems Dynamics Research and SPring-8 Center, Japan
  6. JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Japan
  7. Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Japan
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Cite this article as: eLife 2020;9:e56862 doi: 10.7554/eLife.56862

Abstract

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.

Introduction

Rotary ATPase/ATP synthases, roughly classified into F type and V type ATPases, are marvelous, tiny rotary machines (Yokoyama and Imamura, 2005; Kinosita, 2012; Forgac, 2007; Yoshida et al., 2001; Kühlbrandt, 2019). These rotary motor proteins share a basic molecular architecture composed of a central rotor complex and surrounding stator apparatus. These proteins function to couple ATP hydrolysis/synthesis in the hydrophilic F1/V1 moiety with proton translocation through the membrane embedded hydrophobic Fo/Vo moiety by rotation of the central rotor complex relative to the surrounding stator apparatus, via a rotary catalytic mechanism (Figure 1Kinosita, 2012; Forgac, 2007; Yoshida et al., 2001; Kühlbrandt, 2019; Guo and Rubinstein, 2018).

Figure 1 with 1 supplement see all
Schematic of rotary ATPase/synthases and the rotary catalytic mechanism.

(A) Bacterial FoF1, (B) yeast V-ATPase, (C) Tth V/A-ATPase, (D) a schematic model of the rotary catalytic mechanism. The subunits of the central rotor complex are colored as follows: c-ring, dark blue; a-subunit, red; central axis, purple and cyan; and d-subunit, green.

Thus, both F and V type ATPases are basically capable of either ATP synthesis coupled to the proton motive force (pmf) driven by the membrane potential or proton pumping powered by ATP hydrolysis. The F type ATPase (F-ATPase, or FoF1) in mitochondria functions as an ATP synthase coupled to respiration, whilst in some bacteria the complex can function as an ATP dependent proton pump (Shibata et al., 1992; Kullen and Klaenhammer, 1999).

The V type ATPase (V-ATPase, or VoV1) resides mainly in the membranes of acidic vesicles in eukaryote cells, functioning as a proton pump using a rotary catalytic mechanism (Forgac, 2007; Yokoyama et al., 2003; Imamura et al., 2003). Eukaryotic V-ATPases probably evolved from the prokaryotic enzymes (Gogarten et al., 1989; Tsutsumi et al., 1991), termed Archaeal ATPases or V/A-ATPases (Forgac, 2007; Kühlbrandt and Davies, 2016). The V/A-ATPase from a thermophilic bacterium, Thermus thermophilus (Tth V/A-ATPase) is a rotary ATPase that has been well characterized using both structure and single molecular observation studies (Yokoyama and Imamura, 2005; Yokoyama et al., 2003; Imamura et al., 2003; Iwata et al., 2004; Makyio et al., 2005; Toei et al., 2007; Nakanishi et al., 2018; Schep et al., 2016). The overall structure of Tth V/A-ATPase closely resembles that of the eukaryotic V-ATPase although it lacks some of the accessary subunits of the eukaryotic enzyme (Figure 1B,C). The Tth V1 moiety is composed of four subunits with a stoichiometry of A3B3D1F1 and it is responsible for ATP synthesis or hydrolysis (Yokoyama et al., 1998; Yokoyama et al., 1990). Upon dissociation from Vo, the isolated V1 moiety displays only ATP hydrolysis activity accompanied by rotation of the DF shaft. The Tth Vo moiety, responsible for proton translocation across the membrane, contains a central rotor complex (d1c12) and stator apparatus made up of the a subunit and two EG peripheral stalks (a1E2G2). In the holo Tth V/A-ATPase, pmf drives rotation of the d1c12 rotor complex relative to the surrounding stator, resulting in rotation of the entire central rotor complex (D1F1d1c12) and inducing sequential conformation changes in the A3B3 catalytic hexamer to produce three ATP molecules from ADP and inorganic phosphates per one rotation (Figure 1D).

The eukaryotic V-ATPase is regulated by a unique mechanism involving dissociation/association of V1, that is likely to be a key factor in controlling the pH of acidic vesicles (Kane and Parra, 2000; Sharma et al., 2019; Toei et al., 2010). In yeast, glucose depletion in the culture medium induces dissociation of the V1 domain from Vo domain, resulting in reduced proton pumping activity of the V-ATPase (Figure 1—figure supplement 1A). It is likely that dissociated Vo loses the ability to translocate protons as a result of auto-inhibition (Couoh-Cardel et al., 2015; Qi and Forgac, 2008). In the structure of dissociated yeast Vo, the hydrophilic region of the a subunit (asol) changes its conformation to prevent rotation of the rotor complex (Roh et al., 2018; Mazhab-Jafari et al., 2016). The yeast asol region lies in close proximity to the d subunit, the rotor region of the isolated yeast Vo component. Both the asol region and d subunit represent the trademarks of the V-ATPase family lacking in F-ATPases (see Figure 1A–CIwata et al., 2004). Thus, the asol region and the d subunit appear to be crucial in stabilizing the auto-inhibition structure of the dissociated Vo domain.

A regulatory dissociation/association mechanism has not been reported for the bacterial V/A-ATPase, however, reconstitution experiments suggest an assembly pathway for the holo complex, in which cytosolic V1 associates with membrane Vo (Figure 1—figure supplement 1BKishikawa and Yokoyama, 2012). Thus, proton leak through the Vo domain in Tth membranes may also be blocked by an autoinhibition mechanism similar to that in the eukaryotic enzyme. Indeed, the Tth V/A-ATPase and eukaryotic V-ATPase share very similar structures with Vo moieties comprising the a and d subunits in addition to the c ring.

Structural analysis of several subunits and subcomplexes of V/A-ATPases has been successfully carried out (Iwata et al., 2004; Makyio et al., 2005; Toei et al., 2007; Nagamatsu et al., 2013; Murata et al., 2005). Recent advances of single particle cryogenic microscopy (cryoEM) have facilitated structural analysis of the entire holo complexes of prokaryotic and eukaryotic V-ATPases in several rotational states (Nakanishi et al., 2018; Zhou and Sazanov, 2019; Vasanthakumar et al., 2019). While several structures of the isolated yeast Vo have been reported (Roh et al., 2018; Mazhab-Jafari et al., 2016; Vasanthakumar et al., 2019), a high resolution structure of the isolated Tth Vo is currently unavailable, limiting understanding of the mechanism of enzyme inhibition.

Here, we report a cryoEM structure of isolated Tth Vo at 3.9 Å resolution. The Vo structure reveals that the asol region and d subunit adopt distinct conformation, appropriate for inhibiting rotation of d1c12 relative to the stator a subunit. This conformation is different from that seen in the Vo moiety of the complete Tth V/A-ATPase. Biochemical analysis using Tth Vo reconstituted into liposomes supports inhibition of proton conductance of isolated Vo with a threshold membrane potential. Our results indicate that bacterial and eukaryotic Vo domains use a similar mechanism for auto-inhibition of proton conductance. This mechanism prevents proton leak from Tth cells through an intermediate assembly of the Vo domain of holo V/A-ATPase under physiological conditions.

Results

CryoEM structures of the isolated Vo domain and holo Tth V/A-ATPase

We purified both the Tth V/A-ATPase and Vo domain with a His3-tagged c subunit from membranes of T. thermophilus cells using Ni-NTA resin. The purified complexes were reconstituted into nanodiscs composed of the membrane scaffold protein MSP1E3D1 and POPC lipids. For the Tth V/A-ATPase, acquisition of micrographs was carried out using the Titan Krios electron microscope equipped with a Falcon II direct electron detector. Cryo-EM micrographs of the complexes reconstituted into nanodiscs resulted in higher resolution EM maps compared to those previously reported for the LMNG solubilized preparations (Nakanishi et al., 2018). The strategy of single particle analysis for the Tth V/A-ATPase is summarized in Figure 2—figure supplement 1. We reconstructed the 3D structure of the holo complex rotational state 1 using 71,196 polished single particle images. The final structure of the state one has an overall resolution of 3.6 Å (Figure 2A). After subtracting the EM density of the membrane embedded domain from the density of the whole complex, we obtained a focused density map of A3B3D1F1d1 with two EG peripheral stalks and the soluble arm domain of the a subunit (asol) at 3.5 Å resolution (Figure 2—figure supplement 5). This map allowed us to build an atomic model of A3B3D1F1 (V1) (Figure 2—figure supplement 6). In our map, the obvious density of ADP-Mg was observed in the closed catalytic site, but not clearly observed in the semi-closed site, in contrast to our previously reported structure of the state 1 (PDBID: 5Y5Y). The secondary ADP in the semi-closed site shows a lower occupancy due to low affinity of the semi-closed site for the nucleotide and partial flexibility of the complex (Figure 2—figure supplement 2A). In the recent cryoEM map of Tth V/A-ATPase (PDBID: 6QUM), clear densities, likely corresponding to ADP, were observed in the cavities of the crown-like structure formed by the six β barrel domains of A3B3 (Zhou and Sazanov, 2019). In contrast, these densities were not clearly visible in our structure (Figure 2—figure supplement 2B). This dissimilarity can presumably be explained by differences in the purification procedures; we purified the His-tagged Tth V/A-ATPase using a nickel column, while the authors of the previous study isolated their Tth V/A-ATPase without an affinity purification step.

Figure 2 with 7 supplements see all
EM density map of the enzyme complex.

(A) The holo Tth V/A-ATPase (left) and the focused refined map of A3B3DFd(EG)2asol (right). (B) The isolated Vo domain. Densities corresponding to the individual subunits are colored as follows: A, magenta; B, yellow; D, purple; F, cyan; E and G, gray; a, red; d, green; and c, dark blue. Scale bar = 30 Å.

The purified Vo domain reconstituted into nanodiscs was subjected to single particle analysis using a cryoEM (CRYOARM200, JEOL) equipped with a K2 summit electron direct detector in electron counting mode. The strategy of single particle analysis for TthVo is summarized in Figure 2—figure supplement 3. The 2D class averages disclosed the isolated Vo domain with clearly visible transmembrane helices and a hydrophilic domain extending above the integral membrane region (Figure 2—figure supplement 4). The scaffold proteins and lipids of the nanodiscs surrounding the membrane domain of the isolated Vo were clearly visible. Subsequent 3D classification of the observed Vo states revealed only one major class, indicating that the isolated Vo is structurally homogenous, in contrast to the Tth V/A-ATPase, which was clearly visible in three different rotational states (Nakanishi et al., 2018). Our 3D reconstruction map of the isolated Vo complex was obtained with an overall resolution of 3.9 Å (Figure 2—figure supplement 5). The final map shows clear density for protein components of Vo, including subunit a, subunit d and the c12 ring, but the EM density for both EG stalks, which attach to the asol region, is weak indicating disorder or flexibility in these regions (Figure 2B). In this structure, a C-terminal region of the EG stalk on the distal side is visible. With the exception of these two EG stalks, side-chain densities are detectable for most of the proteins in the complex, allowing construction of a de novo atomic model using Phenix and Coot software (Figure 3A,B, Figure 2—figure supplement 7). The map contains an apparent density inside the c12 rotor ring, likely corresponding to the phospholipids capping the hole of the ring (Figure 3—figure supplement 1A). A further apparent density was identified in the cavity between the a subunit and c12 ring on the upper periplasmic side (Figure 3—figure supplement 1B). This density may also correspond to phospholipids, and we suppose that it functions to plug the cavity between the a subunit and c12 ring, preventing proton leak from the periplasmic proton pathway. Similar densities corresponding to phospholipids were also observed in the recently published cryoEM density map of the holo complex (Zhou and Sazanov, 2019). Notably, the diameter of the c12 rotor ring in the isolated Vo is slightly smaller than that in the Tth V/A-ATPase (Figure 3—figure supplement 2). It is likely that penetration of the short helix of the subunit D into the subunit cavity of subunit d enlarges the diameter of the c12 rotor ring in the Tth V/A-ATPase.

Figure 3 with 2 supplements see all
Atomic model of the isolated Vo domain.

(A) Side view and (B) top view of a-, d-, c-, and EG subunits colored as in Figure 2, respectively. Scale bar represents 30 Å. The proximal and distal subdomains of the a-subunit are circled by dotted lines. (C) Comparison of the relative positions of asol (red) and the d subunit (green) in the isolated Vo domain (left) and the Vo domain in the holo complex (right). Arrows indicate the kinking and twisting points in the asol region of the isolated Vo. Scale bar represents 30 Å. (D) and (E) Specific interactions between the asol region and d subunit at the proximal (D) and distal (E) regions. The regions are specified by black squares in C. Scale bar = 5 Å.

Structure comparison of the isolated and complexed Vo domains

A comparison of structures determined for the isolated Vo domain with that in the holo complex revealed a high degree of similarity in the membrane embedded region. However, there were significant differences in the a subunit. The basic structure of the Tth Vo a subunit is almost identical to the eukaryotic counterpart, comprising a soluble arm domain (asol) and a C-terminal hydrophobic domain responsible for proton translocation via rotation of the c12 ring. The asol region contains two globular α/β folding subdomains responsible for binding of both the proximal and distal EG stalks (Figure 3A and B). Both globular subdomains are connected by a hydrophilic coiled coil with a bent conformation.

In contrast to the Vo structure in the holo complex, the asol region in the isolated Vo is located in close proximity to the d subunit as a result of kinking and twisting of the coiled coil at residues a/L119 and a/A246 (Figure 3C, indicated by the arrows). In this structure, several interactions between the d subunit and asol residues can be observed (Figure 3D). At the proximal site, three amino acid residues, a/E57, a/H65, and a/Q106, form salt bridges or hydrogen bonds with residues d/R38, d/S41, and d/R63 of the d subunit, respectively. Our structure also reveals clearly connected densities between the distal subdomain of the asol region and d subunit (Figure 3E). Four side chains, d/Q138, d/R152, d/R156, and a/R196 apparently form hydrogen bonds with the oxygen atoms in the main chain of a/E201, a/L144, a/A197, and d/R156, respectively. With the exception of the interaction between a/E57 and d/R38 in the proximal site, these interactions are broken by the dynamic movement of asol and conformational changes of the d subunit in the Vo moiety of the holo Tth V/A-ATPase. The conformational changes induced by binding of V1 (A3B3DF) to Vo are described in a separate section below.

Structure of the membrane embedded region of the isolated Vo domain

Our previous low-resolution structure of the Tth V/A-ATPase suggested the involvement of half-channels in proton translocation on both the cytoplasmic and periplasmic sides of the Vo domain (Nakanishi et al., 2018). The atomic model of Vo presented here reveals details of the half-channels formed by the membrane-embedded C-terminal region of the a subunit (aCT) and its interface with the c12 ring. The aCT region contains eight membrane-embedded helices, MH1 to MH8. MH7 and MH8 are the highly-tilted membrane-embedded helices characteristic of rotary ATPases. The cytoplasmic hydrophilic cavity is formed by the cytoplasmic side of MH4, MH5, MH7, and MH8, and the c subunit/chain Z. The cavity is lined by the polar residues, a/R482, a/H491, a/H494, a/E497, a/Y501, a/E550, a/Q554, a/T553, a/H557, and c(Z)/Thr54 (Figure 4A), which make up the cytoplasmic half-channel. The periplasmic sides of MH1, MH2, MH7, and MH8 form the periplasmic hydrophilic cavity, lined with a/D365, a/Y368, a/E426, a/H452, a/R453, a/D455, and c(Y)/E63. The two hydrophilic channels are separated by a salt bridge formed between c(Z)/63Glu, a residue critical for proton translocation, and a/Arg563, a/Arg622, a/Gln619 of MH7 (Figure 4B). This salt bridge is conserved in both eukaryotic and prokaryotic Vo (Mazhab-Jafari et al., 2016; Kishikawa and Yokoyama, 2012). Of note, a salt bridge forms between a single arginine residue and a single glutamic (or aspartic) acid residue in Fo (Kühlbrandt, 2019; Murphy et al., 2019; Guo et al., 2019). Similar to the two-channel model described for other rotary ATPases (Srivastava et al., 2018; Hahn et al., 2018), the two arginine residues on the MH7 and MH8 play an important role in protonation and deprotonation of the carboxy groups on the c12 ring, with the resulting rotation of dc12 driven by proton translocation from the periplasmic to cytoplasmic side (Guo and Rubinstein, 2018; Hahn et al., 2018; Pogoryelov et al., 2010). Notably, in addition to the rigid salt bridge formed between the two a/Arg residues, a/Gln and c/Glu, further interactions between act and the c12 ring are observed. Furthermore, a/Asp392 and Leu393 -c(Y)/Arg49 in the loop region of the c subunit (Figure 4—figure supplement 1A), and the periplasmic sides of MH5 and MH6 are in close proximity to the C-terminal end of the c subunit (Figure 4—figure supplement 1B). These interactions are observed in the Vo moiety of the recently published holo complex structure (Zhou and Sazanov, 2019). Overall, our Vo structure is largely identical to the Vo moiety observed in the holo complex with the exception of some alterations in the hydrophilic domain (Zhou and Sazanov, 2019).

Figure 4 with 1 supplement see all
Structure of the hydrophobic domain of isolated Vo.

(A) The half-channels for proton translocation on both the cytoplasmic and periplasmic sides of the isolated Vo domain. Residues lining the pathways are represented as balls and sticks. Residues from the a-subunit and c-subunit are indicated in red and blue boxes, respectively. Proton flow, as it would occur in the case of ATP synthesis, is represented by arrows. The solid arrows indicate proton flow from the periplasmic side to the c-subunit (green), and from the c-subunit to the cytoplasmic side (blue). The dotted black arrows indicate proton movement due to rotation of the c12-ring. Scale bar = 10 Å. (B) The salt bridge between a/Arg563, Arg622, Gln619 and c/Glu63. Scale bar = 3 Å.

Voltage threshold for proton conductance activity of the isolated Vo domain

Our structure of the isolated Vo domain suggests that the rotation of the c12 rotor ring relative to the stator is mechanically hindered by a defined interaction between the asol region and the d subunit. Previous studies have shown that isolated yeast Vo is impermeable to protons (Couoh-Cardel et al., 2015; Qi and Forgac, 2008), but it was unclear whether proton conductance is also inhibited in the isolated Tth Vo domain. To investigate proton conductance through the isolated Tth Vo, we reconstituted this domain into liposomes energized with a Δψ generated through a potassium ion (K+)/valinomycin diffusion potential. The pH change in the liposomes was monitored with 9-Amino-6-Chloro-2-Methoxyacridine (ACMA); the emission traces at 510 nm excited at 460 nm were recorded (Figure 5). The membrane potential was modulated by varying the external K+ concentration according to the Nernst equation. As shown in Figure 5B, the isolated Vo domain displays no proton conductance when the membrane potential is lower than 120 mV, defining a voltage threshold. The proton conductance through the Vo increases proportionally with the membrane potential when the membrane potential exceeds 130 mV (Figure 5B). The reported membrane potential in bacterial cells varies from −75 to −220 mV depending on growth environment and method of quantification (Lo et al., 2007; Bot and Prodan, 2010). Although the membrane potential of T. thermophilus under physiological conditions is unknown, we reported previously that the Tth V/A-ATPase is capable of ATP synthesis when the membrane potential exceeds −110 mV (Toei et al., 2007). Thus, proton impermeability of the isolated Tth Vo observed at potentials less than −120 mV may function to maintain pmf for ATP synthesis, when Tth Vo exists solely on the cell membrane. In contrast to the Vo domain, several experiments have indicated that proton conductance through the bacterial Fo domain is not sensitive to any specific threshold in membrane potential (Wiedenmann et al., 2008), whereas bacterial FoF1 is sensitive to a membrane potential threshold, likely dependent on the interaction between Fo and F1 (Feniouk et al., 2004). In addition, proton conductance through the Fo domain increases linearly with increasing Δψ loaded on the Fo liposome. These results indicate that there are no or few interactions between the a subunit and c-ring to hinder c-ring rotation in Fo. Together, the observed results suggest that asol of the a subunit and the d subunit, absent from Fo and validated structures of the V type ATPases, can be one of the keys for mechanical inhibition of proton conductance through Vo.

Proton conductance through the isolated Vo domain.

(A) Changes of ACMA fluorescence due to pH changes inside the Vo proteo-liposomes. Values of the membrane potential (ΔΨ) were estimated using the Nernst equation, ΔΨ = RF/zF ln[KCl]o/[KCl]i, as described in the Materials and methods section. (B) The voltage threshold of proton conductance through the Vo domain (mean ± SD, n = 3).

Discussion

The structure of the isolated Tth Vo obtained clearly shows a different conformation from the Vo moiety in the holo-complex. From structural comparison between isolated Vo and the holo complex, it can be suggested that structural changes in isolated Vo observed in two subunits were most likely induced by dissociation of the V1 domain from Vo. In the isolated Vo domain, the d subunit adopts the closed form in which three side chains of the d subunit are able to interact with the distal subdomain of asol (Figure 3E). Once the short helix of the D subunit, an axis subunit of the V1 domain, inserts into the cavity of the d subunit, the interaction between H6 and H11 via d/R90 and d/E195 is broken (Figure 6A and Video 1), resulting in the d subunit adopting an open form, with side chains orientated away from the distal subdomain of asol.

Figure 6 with 2 supplements see all
Conformational changes occurring in both the d- and asol subunits as a result of V1 to Vo binding.

(A) Structural changes in the d subunit caused by insertion of the screw driver helix (SDH). A top view of the d-subunit is shown in the left panel. The d-subunit from the isolated Vo domain and the holo enzyme are colored in green and grey, respectively. Red arrows indicate movements of helices 6–9 (H6–H9). The key helices, H6 and H11, of the d-subunit in the isolated Vo domain and holo complex are shown in panel A, center and right. The H6 helix bends 45° as a result of interaction between the d-subunit and SDH. (B) Structural changes in the distal subdomain of asol. Upon the pivoting movement of asol on the proximal subdomain, the distal subdomain swings 25 Å and twists 15° between the isolated Vo (red) and the holo complex (gray). (C) The EG structure in the distal subdomain of asol (EGd) in the isolated Vo domain (left) and in the holo complex (right). (D) A schematic representation of mechanical inhibition of Vo induced by dissociation of V1. In the isolated Vo domain, rotation of the central rotor is inhibited by interactions between the d-subunit and asol (yellow box, Figure 3D, E).

Video 1
Conformational changes of the d-subunit between isolated Vo and Vo in holo-enzyme.

A morphed movie focuses on the conformational changes of the d-subunit between isolated Vo and Vo in the holo-enzyme. a-, d-, c- and d-subunits are colored in red, green, dark blue, and orange, respectively. d/R90 and d/E195 are represented as balls and sticks. The density map of isolated Vo is shown as a semi-transparent surface.

Another contributing factor is the dynamic motion of the asol region induced by binding the distal EG stalk to the top of the A3B3 from the V1 domain. In the isolated Vo, the N-terminal region of the EG stalk bound to the distal subdomain of asol is at a much steeper angle relative to the horizontal coiled coil structure of asol than that in the holo enzyme (Figure 6B,C and Figure 6—figure supplement 1). This finding suggests that the stalk region also adopts a steep angle, although the stalk and head domain of EG are disordered in the resolved structure and thus not visible in the density map (Figure 2B). Once the C-terminal globular domain of the distal EG stalk binds onto the top of A3B3, the angled distal EG adopts a vertical standing form, resulting in both twisting and kinking of the coiled coil of the hydrophilic arm and the distal globular subdomain of the a subunit (Figure 6C, Video 2). These dynamic motions of asol induce disruption of specific interactions between asol and the d subunit.

Video 2
Conformational changes of a hydrophilic arm of the a-subunit linked to movement of the EG subunits.

A morphed movie focusing on the conformational changes of a hydrophilic arm of the a-subunit between isolated Vo and Vo in the holo-enzyme. EGp and EGd indicate proximal and distal EG subunits. The position of EGd was determined from the low-resolution density map (Figure 6—figure supplement 1). The hydrophilic arm of the a-subunit associated with binding EGd to one of the B subunits from V1, is forced to swing away from the d-subunit, resulting in disruption of the specific interaction between the subunits.

The isolated yeast Vo domain also adopts a conformation where the asol region is in close proximity to the d subunit, resulting in rigid interaction between the stator and rotor that is advantageous for inhibition of proton conductance (Roh et al., 2018; Mazhab-Jafari et al., 2016). Although an atomic model of the yeast holo V-ATPase has yet to be determined, a poly alanine model of the yeast V-ATPase shows that the asol region is some distance away from the d subunit in the Vo moiety (Zhao et al., 2015). In addition, a recently reported structure of the mammal V-ATPase clearly shows that asol is at a distance where it cannot interact with the d subunit (Abbas et al., 2020). This structure suggests that a similar conformational change in Vo is induced by binding of the V1 domain in the yeast V-ATPase, as described by Oot and Wilkins previously (Oot and Wilkens, 2012). Notably, the d subunit in the yeast enzyme differ in conformation between the isolated Vo domain and holo enzyme, in contrast to the Tth enzyme, where the d subunit is in the closed form in the isolated Vo domain (Figure 6—figure supplement 2Vasanthakumar et al., 2019). The d subunit from the mammalian holo V-ATPase adopts a more open conformation than the yeast d subunit from the isolated Vo complex, as seen in the holo Tth V/A-ATPase (Abbas et al., 2020). In addition, Abbas et al. suggest that the d subunit from the yeast holo V-ATPase is also more open compare to that of the yeast isolated Vo (Abbas et al., 2020). These results indicate that the d subunit in the mammalian and yeast V-ATPase also exhibits a conformational change between isolated Vo and holo enzyme.

However, Qi et al. reported that yeast Vo was impermeable to proton even in the absence of interactions between the a-subunit and d-subunit (Couoh-Cardel et al., 2015; Qi and Forgac, 2008). These findings suggest that the interactions between asol and d-subunit are not the only mechanism by which proton permeability is inhibited. In fact, salt bridges between the arginine residues (a/R563, R622 in Tth Vo, a/R735, 799 in yeast Vo) and the glutamate residue (c/E63 in Tth Vo, c/E108 in yeast Vo) (Roh et al., 2018; Mazhab-Jafari et al., 2016; Vasanthakumar et al., 2019) are identified in isolated Vo from both T. thermophilus and yeast Vo. These salt bridges between the stator a subunit and the rotor c-ring inhibit proton permeability by hindering c-ring rotation (Mazhab-Jafari et al., 2016). It is still controversial whether the formation of this salt bridge represents a bona fide process of proton translocation that links deprotonation and re-protonation of glutamate residues in the c subunits. (Kühlbrandt, 2019; Roh et al., 2018; Zhou and Sazanov, 2019; Abbas et al., 2020; Krah et al., 2019; Pierson et al., 2018; Symersky et al., 2012). Undoubtedly, the salt bridge must be broken by both rotation of the c-ring driven by pmf and ATP hydrolysis in V1 in order to perform the functions of ATP synthesis or proton pumping (Figure 5Roh et al., 2018; Vasanthakumar et al., 2019; Abbas et al., 2020).

As described above, eukaryotic and prokaryotic V/A-ATPases appear to share a similar mechanism of conformational change at the Vo moiety, advantageous for preventing proton leakage from cells or acidic vesicles. Nevertheless, there exist some interactions unique to Tth Vo, as described in this paper (Figure 4—figure supplement 1), and to yeast Vo, as reported by previous studies (Roh et al., 2018). This suggests that the auto-inhibition mechanisms of Vo have been conserved during evolution of V type ATPases.

In the isolated yeast Vo, the luminal half-channel, which releases translocated protons to the lumen of acidic vesicles is closed and it is assumed to open transiently during catalysis (Mazhab-Jafari et al., 2016; Krah et al., 2019). In the case of Tth V/A-ATPase, both sides of the half-channel are open (Zhou and Sazanov, 2019). The membrane domain of the a subunit from the isolated Tth Vo is largely identical to that of the holo enzyme (r. m. s. d. = 0.82 Å for A327-E637 of the a subunit), thus the half-channels are likely also open in Tth Vo as observed in the holo enzyme. This indicates that TthVo is more proton permeable than yeast Vo. This difference might be a consequence of the differences between the protein acting as an ATP synthase (Tth enzyme) and ATP driven proton pump (yeast enzyme), as previously suggested (Krah et al., 2019). Further studies, such as computational MD simulation, are required to assess the extent of contribution of each interaction to the auto-inhibition mechanism of Tth Vo.

Our structure of the isolated Vo domain further reveals the mechanism of mechanical inhibition of rotation of the dc12 rotor complex due to strong interactions between asol and the d subunit, regions unique to V-ATPases. These interactions stabilize the isolated Vo domain and protect against loss of the d-subunit in the absence of the rotor-stator interactions mediated by V1 in the holo enzyme (Ediger et al., 2009). This stabilization of Vo is likely to be a key factor for both assembly of holo V-type ATPase complexes and regulation of the eukaryotic V-ATPase via dissociation of V1 from Vo.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background
T. thermophilus
HB8Tamakoshi et al., 1997
Chemical compound, drug14:0 PC (DMPC)Avanti polar lipid850345
Chemical compound, drug16:0-18:1 PC (POPC)Avanti polar lipid850457
Chemical compound, drugn-Dodecyl-beta-D-maltopyranosidecosmo bioD-1304
Chemical compound, drugBiobeads SM-2bio-rad1523920
Chemical compound, drugL-α-Phosphatidylcholine from soybean, Type II-SMerckP5638
Chemical compound, drugn-octyl-β-D-glucosidesigma aldrich850511P
Chemical compound, drug9-Amino-6-Chloro-2-MethoxyacridineThermo FisherA1324
Chemical compound, drugCarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazonesigma aldrichC2920
Chemical compound, drugValinomycinsigma aldrichV0627
Software, algorithmRELIONZivanov et al., 2018RRID:SCR_016274
Software, algorithmMotionCor2Zheng et al., 2017RRID:SCR_016499
Software, algorithmGctfZhang, 2016RRID:SCR_016500
Software, algorithmCOOThttp://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/RRID:SCR_014222
Software, algorithmPhenixhttps://www.phenix-online.org/RRID:SCR_014224
Software, algorithmMolProbityhttp://molprobity.biochem.duke.eduRRID:SCR_014226

Protein preparation

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T. thermophilus V/A-ATPase was expressed with a His3 tag on the C-terminus of the c-subunit using a modified operon generated by the integration vector system (Tamakoshi et al., 1997). Purification of His-tagged Vo was carried out as described previously (Nakanishi et al., 2015). Briefly, membranes of T. thermophilus were suspended in a buffer containing 10% Triton X-100 and sonicated to solubilize membrane proteins. After ultracentrifugation, the supernatant containing V/A-ATPase was applied to a Ni-NTA column. The fractions containing V/A-ATPase were dialyzed against 20 mM Tris-HCl (pH 8.0), 1 mM EDTA for 2 days at 4°C. The combined fraction was applied to a Resource Q column. Eluted fractions were analyzed by SDS-PAGE and the fractions containing Vo and V/A-ATPase were concentrated separately using Amicon 100K molecular weight cut-off filters (Millipore).

For nanodisc incorporation, 25 mM DMPC (Avanti) solubilized in 5% DDM was used. The concentrated Vo fraction, the scaffold protein MSP1E3D1, and DMPC were mixed in a 1:12:600 molar ratio and incubated for 30 min at room temperature. Then, 200 μL of Bio Beads SM-2 equilibrated with wash buffer (20 mM Tris-HCl, pH8.0, 150 mM NaCl) were added into 500 μL of the protein-lipid mixture. After 2 hr of incubation at 4°C with gentle stirring, an additional 300 μL of Bio Beads was added to the mixture prior to overnight incubation at 4°C to form nanodisc. The supernatant of the mixture containing the nanodisc-Vo was applied to a Superdex200 Increase 10/300 column equilibrated in wash buffer. Individual fractions were analyzed by SDS-PAGE and concentrated to ~4 mg/mL. The prepared nanodisc-Vo was stored at 4°C and used for cryo-grid preparation within several days.

V/A-ATPase was reconstituted into lipid nanodiscs using the same protocol as that for Vo, except that 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti) was used as the lipid during reconstitution. Purified V/A-ATPase solubilized in 0.03% n-Dodecyl-β-D-maltoside (DDM) was mixed with the lipid stock and membrane scaffold protein MSP1E3D1 (Sigma) at a specific molar ratio of V/A-ATPase: MSP: POPC lipid = 1: 4: 520 and incubated on ice for 0.5 hr. 200 μl of Bio-beads SM-2 were added to initiate the reconstitution by removing detergents from the system and the mixture was incubated at 4°C for 3 hr with constant rotation. The bio-beads were removed and the nanodisc mixture applied to a Superdex200 Increase 10/300 column (GE Healthcare) pre-equilibrated in buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM MgCl2). Reconstitution was assessed by both size exclusion chromatography and SDS-PAGE. The peak corresponding to the nanodisc-reconstituted V/A-ATPase was immediately used for cryo-EM observation.

Biochemical analysis

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For measurements of proton channel activity, purified Vo was reconstituted into liposomes. L-α-Phosphatidylcholine Type II-S (Sigma-Adrich) was washed repeatedly beforehand to eliminate contamination of K+ ions (Soga et al., 2012), and the L-α-Phosphatidylcholine suspension was adjusted to a final concentration of 40 mg/mL in 4 mM Tricin, and 5 mM MgCl2 . To 250 μL of L-α-Phosphatidylcholine suspension, 250 μL of a solution containing 8% (w/v) n-octyl-β-D-glucoside (Sigma), and 500 mM KCl were added. Then, 60–80 ng of purified Vo was added. After a 30 min incubation at 4°C, 200 μL of Bio Beads SM-2, pre-equilibrated in 2 mM Tricin and 2.5 mM MgCl2, were added to the mixture. The bead mixture was gently stirred for 30 min at room temperature. After that, 300 μL of Bio Beads were added to the mixture and incubated for another 2 hr. The supernatant was ultracentrifuged (40 k rpm, 4°C, 30 min) to remove contaminating KCl. The pellet containing reconstituted proteoliposome was re-suspended in 2 mM Tricin and 2.5 mM MgCl2. The proteoliposomes were used for proton channel assay immediately. Proton channel activity was detected by the fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) (Thermo Fisher), which changes fluorescence in response to pH reduction inside the proteoliposome. Fluorescence changes were monitored using a spectrofluorometer (FP-6200, JASCO). A 1200 μL aliquot of reaction buffer (2 mM Tricin, pH8.0, 2.5 mM MgCl2, 500 mM KCl + NaCl, 1 μL of 30 mg/mL ACMA, 20 μL of proteoliposome) were incubated at 25°C. Proton channel activity was initiated by injection of 1 μL of 0.1 mg/mL valinomycin at the time = 50 s. After 100 s, 1 μL of 0.2 mg/mL carbonyl cyanide-p-trifluoromethoxyphenylhydrazone was added. The initial rate of pH change was estimated from the linear fitting of the initial decay of fluorescence. The membrane potential (ΔΨ) across the liposome membrane was calculated by the Nernst equation, ΔΨ = (kBT/zF)ln([K+]out/[K+]in)=59.2 log([K+]out/[K+]in) in mV at 25°C, where [K+]out was taken to be that of the reaction buffer, and [K+]in was 500 mM as in the buffer for proteoliopsome reconstruction.

Protein concentrations of Vo were determined from UV absorbances calibrated by quantitative amino acid analysis; 1 mg/ml gave an optical density of 0.56 at 280 nm. Polyacrylamide gel electrophoresis in the presence of SDS or AES was carried out as described previously (Nakano et al., 2008). The proteins were stained with Coomassie Brilliant Blue.

EM imaging

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For cryo-grid preparation, Quanfifoil R1.2/1.3 molybdenum grids were glow discharged by an Ion Bombarder (Vacuum Device) for 1 min. 2.4–2.7 μL of nanodisc-Vo were loaded onto the grid and blotted for 9 s with a blot force of 10, wait time of 0 s at 4°C, and 100% humidity using a Vitrobot (FEI). Then, the grid was plunged into liquid ethane without drain time. Cryo-EM movie collection was performed with the CRYOARM200 (JEOL) operating at 200 keV accelerating voltage and equipped with a direct electron detector, K2 Summit (Gatan) in electron counting mode using the data collection software JADAS. The pixel size was 1.1 Å/pix (x5,0000), a total dose of 79.2 e-/ Å2 (1.32 e-/ Å2/frame) with a 12 s exposure time (60 frames), and a defocus range of −1.0 to −3.5 μm.

For V/A-ATPase analysis, gold grids were used to reduce beam-induced movement (Russo and Passmore, 2014). A 2.4 μL aliquot of V/A-ATPase sample at 3.5 mg/ml was added to a 1.2 μm hole, 1.3 μm spacing holey gold grid (Quantifoil UltrAuFoil) in a semi-automated vitrification device (Vitrobot, FEI/Thermo Fisher) at 100% humidity, 4°C. The grid was then automatically blotted once from both sides with filter paper for a 9 s blot time. The grid was then plunged into liquid ethane without a delay time. Preparations of the V/A-ATPase were observed with a Titan Krios (FEI/Thermo Fisher) operating at 300 kV acceleration voltage and equipped with a Falcon II (FEI/Thermo Fisher) detector at a magnification of 75,000x with a pixel size of 1.1 Å, set up to capture 34 frames, corresponding to a total dose of 91 e-/ Å−2 in a defocus range of −2.4 to −3.0 μm.

Image processing

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Image processing was performed using the Relion 3.0.7 software (Zivanov et al., 2018). A total of 5988 cryo-EM movies were collected for isolated Vo and 3694 movies collected for V/A-ATPase. All images were subjected to motion correction using the MotionCor2 program (Zheng et al., 2017) followed by contrast transfer function (CTF) estimation using Gctf (Zhang, 2016). Manual selection of the motion-corrected micrographs results in 3268 good isolated Vo micrographs and 3084 good V/A-ATPase micrographs. For Vo, a template for particle auto-picking was generated by 2D classification of particles picked by the LoG (Laplacian of Gaussian) method implemented in the Relion software, while particles were picked manually to generate references for auto-picking for V/A-ATPase. Vo and V/A-ATPase particles were picked from each selected micrograph by template-based auto-picking and classified by several rounds of reference-free 2D classification (3.14 × 106 and 0.35 × 106 particles images, respectively). After 2D classifications, 706,617 particles selected for Vo and 147,292 particles selected for V/A-ATPase were subjected to several rounds of 3D classification, respectively. The initial model of Vo was generated from the Vo domain of our previous T. thermophilus V/A-ATPase structure (Nakanishi et al., 2018) using UCSF chimera (Pettersen et al., 2004). A total of 175,930 particles selected for Vo and 71,196 particles selected for V/A-ATPase assigned into good 3D classes were subjected to 3D auto-refinement followed by CTF refinement of Bayesian polishing. Then, 157,618 Vo particles were selected from the polished particles by 2D classification. Another round of 3D auto-refine, CTF refinement, and a final round of masked auto-refine gave a Vo map at 3.9 Å resolution and a V/A-ATPase map at 3.6 Å resolution. The resolution was estimated based on the gold standard FSC = 0.143 criterion. However, while the membrane domain was visible it was not well refined in the V/A-ATPase map. This is likely to be due to the structural flexibility between the Vo and V1 domains in this class. Therefore, focused classification with signal subtraction of the membrane domain was carried out for the V/A-ATPase map to obtain high-quality maps and this gave a near-atomic resolution (3.5 Å resolution) map of the hydrophilic domain (A3B3DFE2G2dasol).

Model building

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To generate an atomic model for the isolated Vo domain, each subunit of the Vo complex from the previous structure of the T. thermophilus V/A-ATPase (PDBID: 5Y5X) was fitted into the density map as a rigid body. Notably the a-subunit was divided into soluble and transmembrane domains and these domains fitted into the map separately. The rigid body structures were fitted against the density map manually using the COOT software (Emsley et al., 2010). Then, the manually fitted structures were refined using the phenix.real_space_refine program contained in the Phenix suite software (Adams et al., 2010). These processes were performed over several rounds. The geometry of the atomic model built in this study was checked using the MolProbity tool (Table 1Chen et al., 2010).

Table 1
CryoEM data collection, refinement and model statistics.
TthV/A-ATPaseV1EGdasolIsolated Vo
Data collection
Electron microscopeTitan KriosCRYOARM200
Electron detectorFalcon IIK2 summit
Magnification75,00050,000
Voltage (kV)300200
Electron exposure (e-2)9179.2
Defocus range (μm)2.4–3.01.0–3.0
Pixel size (Å)1.11.1
Movie No.36945988
Frame per movie3460
Automation softwareEPUJADAS
Data processing
Total extracted particles3.5 × 1053.14 × 106
Total particle after 2D144,758706,617
Resolution (Å)3.63.53,93
Sharpening B-factor−81.07−60.25−110.87
EMDB ID300133001430015
Model building and refinement
Initial models-5Y5Y5Y5X, 1V9M
Building and refinement package-COOT, phenixCOOT, phenix
Total atom No.-26,63113,888
Total residue No.-34181894
Total chain No.-816
Ligands-ADP-
cc_mask-0.850.82
Ramachandran favored-88.89%92.75%
Ramachandran outliers-0.03%0.00%
Rotamer outliers-11.45%0.37%
c-beta deviation-00
CaBLAM outliers-5.94%2.84%
Clashscore-6.3820.01
RMSD bonds (Å)-0.0060.006
RMS angle (°)-0.6690.725
MolProbity score-2.732.26
PDB ID-6LY86LY9

Part of the 4.7 Å resolution hydrophilic domain structure of the T. thermophilus V/A-ATPase (PDBID: 5Y5Y) was fitted into the map of A3B3DFE2G2dasol. A rough initial model was refined against the map with the Phenix suite phenix.real_space_refine program (Adams et al., 2010). The initial model was extensively manually corrected residue by residue in the COOT graphics program (Emsley et al., 2010), in particular with respect to side-chain conformations. The peripheral stalks and d-subunit were removed because of a low resolution in these regions. The corrected model was again refined by the phenix.real_space_refine program with secondary structure, and the resulting model manually checked by COOT (Emsley et al., 2010). This iterative process was performed for multiple rounds to correct any remaining errors until the model was in good agreement with the geometry, as reflected by the MolProbity score of 2.21 for isolated Vo and 2.75 for A3B3DFE2G2dasol .

For model validation against over-fitting, the built models were used for calculation of FSC curves against both half maps, and those were compared with the FSCs of the final models against the final density maps used for model building by the phenix.refine program .

References

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    Thermus thermophilus membrane-associated ATPase indication of a eubacterial V-type ATPase
    1. K Yokoyama
    2. T Oshima
    3. M Yoshida
    (1990)
    The Journal of Biological Chemistry 265:21946–21950.
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Decision letter

  1. Olga Boudker
    Senior Editor; Weill Cornell Medicine, United States
  2. David M Kramer
    Reviewing Editor; Michigan State University, United States
  3. Geoffry A Davis
    Reviewer; Michigan State University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The work determined Cryo-EM structures of both the intact (holo) and membrane embedded portions of the V-ATPase from Thermus thermophilus and gives new insights and testable models for the structural and mechanism of the auto-inhibition of the complex, a long-standing open question.

Decision letter after peer review:

Thank you for submitting your article "The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Olga Boudker as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Geoffry A. Davis (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

The work determined Cryo-EM structures of both the intact (holo) and membrane embedded portions of the V-ATPase from Thermus thermophilus in an attempt to understand the auto-inhibition of the membrane embedded V0. Analysis of structural differences between these forms enzyme support an interesting structural and functional mechanism for the auto-inhibition of V0.

Revisions for this paper:

Both reviews found the work to be exciting, but that some changes were needed. Their general consensus was that it should be possible to address these issues without additional experimental data, by deeper analyses of the data set already presented, together with a reworking of the Discussion. Reviewer 1 points out that the yeast V1V0 structure does not, apparently, show the same structural changes attributed in this work to auto-inhibition, and some discussion of this possible discrepancy, or alternative mechanism, is needed. A related point was brought up by reviewer 2, (major comments 2 and 3) asking for a clearer comparison between the mechanistic implications of the current and yeast structures, in particular addressing the possible structural changes associated with the catalytic cycle, assembly and auto-inhibition. A deeper and broader discussion that addresses these issues should make the paper have a wider impact.

Reviewer #1:

The manuscript by Kishikawa et al. determines the structures of both the intact (holo) and membrane-embedded portions of the V-ATPase from Thermus thermophilus in an attempt to understand the auto-inhibition of the membrane-embedded V0. Using Cryo-EM to determine the two structures, the authors have identified structural differences between the holo enzyme and isolated V0, specifically in relation to the a- and d-subunit locations, leading to their proposal that this conformational shift induces the auto-inhibition of V0 through the formation of a tighter V0 complex and side chain interactions that become possible with removal/loss of V1. The data and discussion provide an interesting structural model for the auto-inhibition of V0 and provide a framework for testing the model.

Major comments:

From the Discussion, the authors say that the isolated V0 structure "clearly shows the inhibitory mechanism for preventing H+ conductance." While the structural differences they present do suggest that the conformational changes in the a- and d- subunits are likely the major mechanism of auto-inhibition, it is not necessarily clearly defined, and their following sentence uses the phrase "most likely" which is a more appropriate description of their model. While the authors present a reasonable model for the structural changes seen between the holo V-ATPase and the isolated V0 that could lead to mechanical changes (Figure 6), clear evidence that these structural changes lead to auto-inhibition are not presented.

The differences between the T. thermophilus and yeast V-ATPase structures require more discussion than provided . The mechanism of auto-inhibition proposed by the authors is primarily the conformational changes between subunits a and d. However, the yeast V0 structure does not show changes in subunit d (Figure 6—figure supplement 2) and the yeast V0 structure showed no differences when subunit d was not present (Mazhab-Jafari et al., 2016), leading the authors of the yeast structure to state: "Consequently, the movement of the N-terminal domain of subunit a, and inhibition of proton translocation, cannot be due to interaction of the N-terminal domain of subunit a with subunit d." While the yeast V1V0 structure has not been solved, and the subunit composition differences between yeast and T. thermophilus may also be involved in structural changes, it is important to adequately address this incongruence between observed datasets and the model proposed in the manuscript as it is stated as a conserved model for V-ATPases.

Figure 4 nicely shows a membrane potential activation threshold for isolated V0. However, the results of this experiment are not fully addressed in the text.

The authors cite membrane potentials measured in E. coli (-75-140 mV), although higher potentials have also been proposed (ex: -220mV, Bot and Prodan, 2010). While it is unclear what potential is biologically relevant for T. thermophilus V-ATPase, the results in Figure 4 should be addressed in relation to what they mean for auto-inhibition of V0, as their preparations appear to be capable of proton pumping at greater than ~ 120 mV driving potential. Previous work with other (F-type) ATPases have also shown voltage gating, likely dependent upon F1 and F0 interactions (Feniouk et al., 2004). The voltage threshold measured for isolated V0 is similar to the threshold required to synthesize ATP in the holo enzyme (~110 mV) measured by the same group (Toei et al., 2007). It is therefore unclear from the results of Figure 4 why the experiment was performed and what the results mean relating to the auto-inhibition of V0.

Reviewer #2:

Kishikawa et al. report in the manuscript entitled "The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance" the molecular basis of closure of the V/A-type ATPase from Thermus thermophilus. The authors solve the V1Vo complex ("holo V/A-ATPase") and the autoinhibited Vo domain (in absence of V1). They observe a different conformation of the N-terminal domain of subunit a ("asol") in either states. In addition, they carry out proton conductance experiments of the isolated Vo domain, confirming that the isolated Vo domain is not able to translocate protons under physiological conditions. I think that the data described in this manuscript are important and should be considered for publication in eLife after substantial revisions. The authors should add additional and revise parts of their analysis (comparison of the Vo domains in the holo and isolated state) and take into account previously published literature on eukaryotic V-type ATPases, compare these data with the results obtained and draw general conclusions by these comparisons.

General comments:

English should be slightly improved throughout the whole manuscript. The paper is not well referenced, as the authors omit references in many places again to link directly to the relevant literature, which makes an evaluation complicated, as again the corresponding reference must be looked up. Furthermore, the authors neglect relevant literature. For example, they write "It is likely that the dissociated Vo loses the ability to translocate protons as a result of auto-inhibition." but do not refer to the relevant reference (Zhang et al., JBC, 1992; https://www.jbc.org/content/267/14/9773.short). Furthermore, previously released structural (e.g. Murata et al, 2005, Murata et al., PNAS, 2011, Abbas et al., 2020, Vasanthakumar et al., 2019, Tani et al., Microscopy, 2013) and computational (e.g. Krah et al., 2019) data on the V-ATPase are neglected, as well as early observations in evolutionary related F-type ATP synthases (e.g. Pogoryelov et al.,2010 (analogous to Murata, PNAS, 2011), Symersky et al., 2012), discussing possible mechanistic modes of action of proton translocation for these enzymes. It has also been proposed earlier that the N-terminal domain of subunit a adopts an alternative confirmation during the apo and holo state binding to V1 in the micro molar range (Oot and Wilkens, 2012).

Additional major comments:

1) "The side chain of d/R59 1 likely forms π-π stacking with a/R103"

I would expect a repulsion due to the positive charge of both residues, instead of stabilization by "π-π stacking". A closer look into the chemical environment near these residues may be required and re-modelling of the side chains may be necessary.

2) "Our structure of the isolated Vo suggests that the rotation of c12 rotor ring relative to the stator is mechanically hindered by a defined interaction between the asol and d subunit."

The authors suggest that the rotor is not able to rotate because it is "mechanically hindered by a defined interaction between the asol and d subunit" and thus ions (protons) cannot be transducted. However, it should be taken into account that the energy transduction of the catalytic event (hydrolysis) in eukaryotic V1 drives the rotation of the c-ring and the conformational change of the N-terminal domain of subunit a may be caused by assembly/disassembly of Vo and V1 (see e.g. Oot and Wilkens., 2012). In addition, computational data suggest that the c-ring is locked in an ion-locked conformation while the luminal channel (in yeast V-ATPase) is not accessible (Krah et al., 2019), also discussed in the reports of the cryo-EM structures (e.g. Mazhab-Jafari et al., 2016). If this also is likely for V/A-type ATPases, would be needed to be tested (e.g. calculation of the solvent accessible surface area for the periplasmic half-channel in the isolated Vo state; the holo V/A-type ATPase has been proposed to be accessible to water Zhou and Sazanov, 2019).

3) The authors further conclude: "Together, the observed results strongly suggest that the asol of the a subunit and the d subunit, absent in Fo and hallmarks structure of the V type ATPases, are key for mechanical inhibition of proton conductance through Vo.". Together with the above-mentioned previously published data, I think that "asol" and d may have an essential function, but likely involved in assembly of Vo with V1. For further conclusions about mechanistic features, the authors should also calculate the solvent accessible surface area for both half-channels of the final models (isolated Vo and V1Vo complex) to study if the channels are open (accessible to water) or closed (not accessible to water and other protein residues, proton transfer via water wire or side chains (reduced side chain flexibility in a dry environment) unlikely). In addition, the authors should also present other structural differences in Vo (if any) for holo (V1Vo) and isolated Vo state, as e.g. differences at the a/c interface (in addition to the data shown in the SI). If there are differences found in comparison to previously published data on eukaryotic V-type ATPases, these discrepancies should be discussed, taking into account that V/A-type ATPases are also able to synthesize ATP (previous work in the same lab).

4) The Discussion section should be also adapted based on the above said.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance" for further consideration by eLife. Your revised article has been evaluated by Olga Boudker (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Please respond in the Discussion to the comment from reviewer #2, specifically, "…the chemical environment around these residues should be described in detail. Alternatively, this sentence could be removed from the manuscript." The text does not have to fully agree with the reviewer, but should directly address the issue. Also, as noted by reviewer 1, some general editing for spelling/grammar.

Reviewer #1:

The revised manuscript "Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance" by Kishikawa et al. provides structural data for both the holo V1Vo and inhibited Vo ATPase from T. thermophilus and uses structural comparison to attempt to elucidate mechanism(s) to explain autoinhibition of the Vo complex. The authors also use their structural data to compare with other available structures of V ATPases to discuss the structural similarities and differences across organisms.

The authors have presented their findings in a thoughtful, structured manner with a focus on how their findings enhance the greater understanding of this protein complex. While the discussion touches upon the evolutionary differences and significance, they authors have done an admirable job to not over-interpret the data that they have collected, but rather suggest further mechanistic studies that can help support or disprove hypotheses based on their structures. The authors have also improved the manuscript in revision by minimizing repetition of findings, which improves the structure of the manuscript.

Reviewer #2:

The manuscript by Kishikawa et al. "The mechanical inhibition of the isolated Vo from V/A-ATPase for proton conductance" has been significantly improved and most of my concerns have been addressed. The work is important and should be published in eLife. However, there are still two issues which should be discussed/corrected :

1) The authors wrote: "As the reviewer pointed out, the two arginines are generally repulsive to each other. However, as shown in the added references, in a number of cases arginine is known to be involved in the enzymatic function by interacting with each other. Furthermore, our model is supported by comparison with the model reported by Sazanov et al. (PDBID: 6QUM)."

The arginine – arginine interaction is still not sufficiently described. Reference 33 is claiming that in ~90 % of the structures, which show interactions of positively charged residues are stabilized by counter charges (such as carboxylate groups).

I also had a look at the structure of the holo-V/A ATPase (pdb-ID: 6QUM). In this structure two arginine residues (aR563 and aR622) are very near, but stabilized by counter charges of aE627 and cE63. However, in case of residues aR59 and dR103 this close distance cannot be observed in the Sazanov structure (pdb ID: 6QUM). The distance of the guanidinium groups of both residues is larger than 10 Å (no interaction). In addition, in the vicinity of aR59 and aR103 several carboxylate side chains can be found, which may interact with both guanidinium groups. In addition, aR59 and aR103 are at the surface of the protein (pdb-ID: 6QUM) and thus I would expect them to be solvated if not bound to carboxylate groups.

Thus, I still think that the chemical environment around these residues should be described in detail. Alternatively, this sentence could be removed from the manuscript.

https://doi.org/10.7554/eLife.56862.sa1

Author response

Reviewer #1:

[…] Major comments:

From the Discussion, the authors say that the isolated V0 structure "clearly shows the inhibitory mechanism for preventing H+ conductance." While the structural differences they present do suggest that the conformational changes in the a- and d- subunits are likely the major mechanism of auto-inhibition, it is not necessarily clearly defined, and their following sentence uses the phrase "most likely" which is a more appropriate description of their model. While the authors present a reasonable model for the structural changes seen between the holo V-ATPase and the isolated V0 that could lead to mechanical changes (Figure 6), clear evidence that these structural changes lead to auto-inhibition are not presented.

We agree with the reviewer's comments. Our structures and data do not direct evidence that the interaction between a subunit and d subunit is a major factor in the inhibited mechanism in the isolated Tth Vo. Therefore, we rewrote the discussion entirety focusing on structure changes of asol and d subunit induced by association of V1 in the discussion part. Also, we mention not only the interactions between the a and d subunits, but also the interaction between a and c-ring in the membrane embedded region. We also indicated some points that need to be clarified in the future.

Furthermore, we carefully discussed the suggestion that the conformational changes in a subunit and d subunit were common across species and raised the possibility that these conformational change might be a conserved auto-inhibition mechanism.

“The structure of the isolated Tth Vo obtained clearly shows how it adopts a structure different from the Vo moiety in the holo-complex. From structural comparison between isolated Vo and the holo complex, it can be suggested that structural changes in isolated Vo observed in two subunits were most likely induced by dissociation of the V1 domain from Vo.”

“On the other hand, Qi et al. reported that yeast Vo was impermeable to proton even when the interactions between the a-subunit and d-subunit were absent. These findings suggest that the interactions between asol and d-subunit are not the only mechanism by which proton permeability is inhibited. In fact, salt bridges between the arginine residues (a/R563, R622 in Tth Vo, a/R735, 799 in yeast Vo) and the glutamate residue (c/E63 in Tth Vo, c/E108 in yeast Vo) are identified in both isolated Vo from T. thermophilus and yeast Vo, respectively. These salt bridges between the stator a subunit and the rotor c-ring inhibit proton permeability by hindering c-ring rotation. It is still controversial whether the formation of this salt bridge represents a bona fide process of proton translocation that links deprotonation and re-protonation of glutamate residues in the c subunits. Undoubtedly, the salt bridge must be broken by the c-ring rotation driven by pmf across the membrane or ATP hydrolysis in V1 to perform the functions of ATP synthesis or proton pumping (Figure 5).”

“Further studies, such as computational MD simulation, are required to assess the extent of contribution of each interaction to the auto-inhibition mechanism of Tth Vo.”

The differences between the T. thermophilus and yeast V-ATPase structures require more discussion than provided. The mechanism of auto-inhibition proposed by the authors is primarily the conformational changes between subunits a and d. However, the yeast V0 structure does not show changes in subunit d (Figure 6—figure supplement 2) and the yeast V0 structure showed no differences when subunit d was not present (Mazhab-Jafari et al., 2016), leading the authors of the yeast structure to state:

We discussed the differences between Vo from T. thermophilus and eukaryotes in more detail and mentioned V1 induced structural change of d subunit in Discussion section. Please read the changes below and the responses to the first major comment.

“The d subunit from the mammal holo V-ATPase adopts a more open conformation than the yeast d subunit from the isolated Vo complex, as seen in the holoTth V/A-ATPase. In addition, Abbas et al. suggest that the d subunit from yeast holo V-ATPase is also more open. These results indicate that the d subunit in eukaryotic V-ATPase also shows the conformational change between isolated Vo and holo enzyme.”

“As described above, eukaryotic and prokaryotic V/A-ATPases appear to share a similar mechanism of conformational change at the Vo moiety that is advantageous for preventing proton leakage from cells or acidic vesicles. Nevertheless, there exist some interactions unique to Tth Vo, as described in this paper (Figure 4—figure supplement 1), and to yeast Vo, as reported by previous papers.”

"Consequently, the movement of the N-terminal domain of subunit a, and inhibition of proton translocation, cannot be due to interaction of the N-terminal domain of subunit a with subunit d." While the yeast V1V0 structure has not been solved, and the subunit composition differences between yeast and T. thermophilus may also be involved in structural changes, it is important to adequately address this incongruence between observed datasets and the model proposed in the manuscript as it is stated as a conserved model for V-ATPases.

As mentioned in our response to the first comment, we carefully discussed the similarities and the differences between T. thermophilus Vo and yeast Vo. Also see our first response

Figure 4 nicely shows a membrane potential activation threshold for isolated V0. However, the results of this experiment are not fully addressed in the text.

The authors cite membrane potentials measured in E. coli (-75-140 mV), although higher potentials have also been proposed (ex: -220mV, Bot and Prodan, 2010). While it is unclear what potential is biologically relevant for T. thermophilus V-ATPase, the results in Figure 4 should be addressed in relation to what they mean for auto-inhibition of V0, as their preparations appear to be capable of proton pumping at greater than ~ 120 mV driving potential. Previous work with other (F-type) ATPases have also shown voltage gating, likely dependent upon F1 and F0 interactions (Feniouk et al., 2004). The voltage threshold measured for isolated V0 is similar to the threshold required to synthesize ATP in the holo enzyme (~110 mV) measured by the same group (Toei et al., 2007). It is therefore unclear from the results of Figure 4 why the experiment was performed and what the results mean relating to the auto-inhibition of V0.

The structure of Vo determined in this study was similar to that of yeast Vo, suggesting that Tth Vo also adopted similar to the yeast Vo inhibited form for proton conductance. However, the inhibition of proton conductance in isolated Tth Vo had not been reported yet. Considering that the membrane potential of bacteria varies depending on the environment, we focused on the pmf required for ATP synthesis (-110 mV), which value is close to the voltage threshold for proton conductance (-120 mV), and assumed that the voltage threshold is might be important to maintain sufficient pmf for ATP synthesis. The discussion has been expanded in the Results section of the manuscript. The changes are shown in red, so please refer to them.

“Previous studies have shown that isolated yeast Vo shows impermeability to proton, but it was unclear whether proton conductance is also inhibited in the isolated Tth Vo domain.”

“The reported membrane potential in bacterial cells varies from -75 to -220 mV depending on growth environment and methods of quantification. Although the membrane potential of T. thermophilus under physiological conditions is unknown, we reported previously that the Tth V/A-ATPase is capable of ATP synthesis when the membrane potential exceeds -110 mV. Thus, proton impermeability of the isolated Tth Vo observed at the potential less than -120 mV may function to maintain pmf for ATP synthesis, when Tth Vo exists solely on the cell membrane. In contrast to the Vo domain, several experiments have indicated that proton conductance through the bacterial Fo domain does not show any threshold for the membrane potential, whereas bacterial FoF1 displays the threshold likely depending on the interaction between Fo and F1. In addition, proton conductance through the Fo domain increases linearly with increasing Δψ loaded on the Fo liposome. These results indicate that there is no or few interactions between the a subunit and c-ring to hinder c-ring rotation in Fo. Together, the observed results suggest that asol of the a subunit and the d subunit, that are absent from Fo and validated structures of the V type ATPases, can be one of the keys for mechanical inhibition of proton conductance through Vo”

Reviewer #2:

[…] General comments:

English should be slightly improved throughout the whole manuscript. The paper is not well referenced, as the authors omit references in many places again to link directly to the relevant literature, which makes an evaluation complicated, as again the corresponding reference must be looked up. Furthermore, the authors neglect relevant literature. For example, they write "It is likely that the dissociated Vo loses the ability to translocate protons as a result of auto-inhibition." but do not refer to the relevant reference (Zhang et al., JBC, 1992; https://www.jbc.org/content/267/14/9773.short). Furthermore, previously released structural (e.g. Murata et al, 2005, Murata et al., PNAS, 2011, Abbas et al., 2020, Vasanthakumar et al., 2019, Tani et al., Microscopy, 2013) and computational (e.g. Krah et al., 2019) data on the V-ATPase are neglected, as well as early observations in evolutionary related F-type ATP synthases (e.g. Pogoryelov et al., 2010 (analogous to Murata, PNAS, 2011), Symersky et al., 2012), discussing possible mechanistic modes of action of proton translocation for these enzymes. It has also been proposed earlier that the N-terminal domain of subunit a adopts an alternative confirmation during the apo and holo state binding to V1 in the micro molar range (Oot and Wilkens., 2012).

As suggested by the reviewer, we have reviewed the suggested references, then reconsidered the conclusions of our previous manuscript. Especially, we focused on the stator-rotor interaction in the membrane embedded region in relation to the auto-inhibition mechanism of Tth Vo. The changes are described in detail below. The English of the manuscript was proofread by a scientific proofreader throughout.

“Structural analysis of several subunits and subcomplexes of V/A-ATPases has been successfully carried out. Recent advances of single particle cryogenic microscopy (cryoEM) have facilitated structural analysis of the entire holo complexes of prokaryotic and eukaryotic V-ATPases in several rotational states. While the structure of the isolated yeast Vo has been reported in several studies, a high resolution structure of the isolated Tth Vo is still unavailable, limiting understanding the mechanism of enzyme inhibition.”

“Similarly to the two-channel model described for other rotary ATPases, the two arginine residues on the MH7 and MH8 play an important role in protonation and deprotonation of the carboxy groups on the c12 ring, with the resulting rotation of dc12 driven by proton translocation from the periplasmic to cytoplasmic side.”

“In addition, a resently reported structure of the mammal V-ATPase clearly shows that asol is at a distance where it cannot interact with the d subunit. This structure suggests that a similar conformational change in Vo is induced by binding of the V1 domain in the yeast V-ATPase, as described Oot and Wilkins previously.”

“On the other hand, Qi et al. reported that yeast Vo was impermeable to proton even when the interactions between the a-subunit and d-subunit were absent. These findings suggest that the interactions between asol and d-subunit are not the only mechanism by which proton permeability is inhibited. In fact, salt bridges between the arginine residues (a/R563, R622 in Tth Vo, a/R735, 799 in yeast Vo) and the glutamate residue (c/E63 in Tth Vo, c/E108 in yeast Vo) are identified in both isolated Vo from T. thermophilus and yeast Vo, respectively. These salt bridges between the stator a subunit and the rotor c-ring inhibit proton permeability by hindering c-ring rotation. It is still controversial whether the formation of this salt bridge represents a bona fide process of proton translocation that links deprotonation and re-protonation of glutamate residues in the c subunits. Undoubtedly, the salt bridge must be broken by the c-ring rotation driven by pmf across the membrane or ATP hydrolysis in V1 to perform the functions of ATP synthesis or proton pumping (Figure 5)”

Additional major comments:

1) "The side chain of d/R59 1 likely forms π-π stacking with a/R103"

I would expect a repulsion due to the positive charge of both residues, instead of stabilization by "π-π stacking". A closer look into the chemical environment near these residues may be required and re-modelling of the side chains may be necessary.

As the reviewer pointed out, the two arginines are generally repulsive to each other. However, as shown in the added references, in a number of cases arginine is known to be involved in the enzymatic function by interacting with each other. Furthermore, our model is supported by comparison with the model reported by Sazanov et al. (PDBID: 6QUM).

Added reference: Neves, M. A., Yeager, M. and Abagyan, R. Unusual arginine formations in protein function and assembly: rings, strings, and stacks. J. Phys. Chem. B 116, 7006-13 (2012)

2) "Our structure of the isolated Vo suggests that the rotation of c12 rotor ring relative to the stator is mechanically hindered by a defined interaction between the asol and d subunit."

The authors suggest that the rotor is not able to rotate because it is "mechanically hindered by a defined interaction between the asol and d subunit" and thus ions (protons) cannot be transducted. However, it should be taken into account that the energy transduction of the catalytic event (hydrolysis) in eukaryotic V1 drives the rotation of the c-ring and the conformational change of the N-terminal domain of subunit a may be caused by assembly/disassembly of Vo and V1 (see e.g. Oot and Wilkens., 2012).

As the reviewer suggested, we proposed the interactions between asol and d subunit are disrupted by the conformational change induced by binding V1 in both eukaryotic and prokaryotic V-ATPases. This idea is summarized in Figure 6 and the Discussion section. In addition, c-ring rotation driven by ATP hydrolysis in V1 might disrupt the interactions between the a subunit and the c-ring. This is also mentioned in the Discussion section. In the revised manuscript, we carefully discussed the common features and differences between yeast and T. thermophilus Vo. Therefore, the Discussion section has been re-written in line with the reviewer’s suggestions. Please read our response to the General comments and the changes below.

“In addition, a resently reported structure of the mammal V-ATPase clearly shows that asol is at a distance where it cannot interact with the d subunit. This structure suggests that a similar conformational change in Vo is induced by binding of the V1 domain in the yeast V-ATPase, as described Oot and Wilkins previously.”

“The d subunit from the mammal holo V-ATPase adopts a more open conformation than the yeast d subunit from the isolated Vo complex, as seen in the holoTth V/A-ATPase. In addition, Abbas et al. suggest that the d subunit from yeast holo V-ATPase is also more open. These results indicate that the d subunit in eukaryotic V-ATPase also shows the conformational change between isolated Vo and holo enzyme.”

In addition, computational data suggest that the c-ring is locked in an ion-locked conformation while the luminal channel (in yeast V-ATPase) is not accessible (Krah et al., 2019), also discussed in the reports of the cryo-EM structures (e.g. Mazhab-Jafari et al., 2016). If this also is likely for V/A-type ATPases, would be needed to be tested (e.g. calculation of the solvent accessible surface area for the periplasmic half-channel in the isolated Vo state; the holo V/A-type ATPase has been proposed to be accessible to water Zhou and Sazanov, 2019).

The reviewer is right, it has been suggested that in the yeast Vo, the luminal half channel is closed and that the channel opens transiently during the reaction. In contrast, in the prokaryotic V/A-ATPase, it has been suggested that both sides of the half channel are open. The membrane domain of the a subunit, which mainly composes the half channels, is very similar in the isolated Tth Vo to that of holo enzyme (r. m. s. d = 0.82 Å). Therefore, the half channels are mostly open in the isolated Tth Vo. These differences might affect the auto-inhibition of proton translocation. Based on the comparison, we modified the manuscript and stated the issues in the Discussion section as below.

“In the isolated yeast Vo, the luminal half-channel for releasing translocated protons to the lumen of acidic vesicles is closed and it is assumed to open transiently during catalysis. In the case of Tth V/A-ATPase, both sides of the half-channel are not enclosed. The membrane domain of the a subunit from the isolated Tth Vo is mostly identical to that of the holo enzyme (r. m. s. d. = 0.82 Å for A327-E637 of a subunit), thus the half-channels are likely open in Tth Vo as observed in holo enzyme. This indicates that TthVo is more proton permeable than yeast Vo. This difference might be derived from the difference between ATP synthesis and ATP driven proton pump, as suggested. Further studies, such as computational MD simulation, are required to assess the extent of contribution of each interaction to the auto-inhibition mechanism of Tth Vo.”

3) The authors further conclude: "Together, the observed results strongly suggest that the asol of the a subunit and the d subunit, absent in Fo and hallmarks structure of the V type ATPases, are key for mechanical inhibition of proton conductance through Vo.". Together with the above-mentioned previously published data, I think that "asol" and d may have an essential function, but likely involved in assembly of Vo with V1.

As suggested by the reviewer, this sentence ("Together, the observed results……… through Vo."). has been excluded in the revision. In addition, we stated contribution of the salt bridges between the stator a subunit and c-ring into the inhibition of proton conductance. Please refer to our response to the General comments. As mentioned in the last paragraph of our Discussion section, we recognize the importance of asol and d subunit for assembly with V1. however, here we focus on the conformational changes of both asol and d subunit induced by binding of V1 to Vo rather than on their role in the complex assembly.

For further conclusions about mechanistic features, the authors should also calculate the solvent accessible surface area for both half-channels of the final models (isolated Vo and V1Vo complex) to study if the channels are open (accessible to water) or closed (not accessible to water and other protein residues, proton transfer via water wire or side chains (reduced side chain flexibility in a dry environment) unlikely). In addition, the authors should also present other structural differences in Vo (if any) for holo (V1Vo) and isolated Vo state, as e.g. differences at the a/c interface (in addition to the data shown in the SI). If there are differences found in comparison to previously published data on eukaryotic V-type ATPases, these discrepancies should be discussed, taking into account that V/A-type ATPases are also able to synthesize ATP (previous work in the same lab).

As it is indicated by the reviewer, the luminal half-channel is enclosed in the isolated yeast Vo (Mazhab-Jafari et al., 2016, Krah et al., 2019). In contrast, both sides of the half-channel are open in holoTth V/A-ATPase (Zhou and Sazanov, 2019). As mentioned in the Results section (sub section “Structure comparison of the isolated Vo with the holo complex”), there are no obvious dissimilarities except for the minor differences mentioned in the manuscript. Considering the high similarity between the isolated Tth Vo and Vo in holo enzyme, we have concluded that the half-channels are also open in the isolated Tth Vo, and stated this in the Discussion section of the manuscript. Please refer to our answer to the 2) comments.

In addition, we have discussed common features and differences between the prokaryotic and eukaryotic isolated Vo in the Discussion section. The reviewer 2 suggested structural comparisons of the isolated Vo and holo enzymes to discuss the factors that create different functional requirements for ATP synthesis or hydrolysis. However, in our manuscript, the obtained map of Vo domain in the holo enzyme was not sufficient to build an atomic model. Also, the high resolution structure of yeast V-ATPase and mammalian isolated Vo are lacking. Therefore, we would like to withhold the discussion of comparison with the eukaryotic holo enzymes.

4) The Discussion section should be also adapted based on the above said.

We rewrote the Discussion section as mentioned above.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #2:

The manuscript by Kishikawa et al. "The mechanical inhibition of the isolated Vo from V/A-ATPase for proton conductance" has been significantly improved and most of my concerns have been addressed. The work is important and should be published in eLife. However, there are still two issues which should be discussed/corrected :

1) The authors wrote: "As the reviewer pointed out, the two arginines are generally repulsive to each other. However, as shown in the added references, in a number of cases arginine is known to be involved in the enzymatic function by interacting with each other. Furthermore, our model is supported by comparison with the model reported by Sazanov et al. (PDBID: 6QUM)."

The arginine – arginine interaction is still not sufficiently described. Reference 33 is claiming that in ~90 % of the structures, which show interactions of positively charged residues are stabilized by counter charges (such as carboxylate groups).

I also had a look at the structure of the holo-V/A ATPase (pdb-ID: 6QUM). In this structure two arginine residues (aR563 and aR622) are very near, but stabilized by counter charges of aE627 and cE63. However, in case of residues aR59 and dR103 this close distance cannot be observed in the Sazanov structure (pdb ID: 6QUM). The distance of the guanidinium groups of both residues is larger than 10 Å (no interaction). In addition, in the vicinity of aR59 and aR103 several carboxylate side chains can be found, which may interact with both guanidinium groups. In addition, aR59 and aR103 are at the surface of the protein (pdb-ID: 6QUM) and thus I would expect them to be solvated if not bound to carboxylate groups.

Thus, I still think that the chemical environment around these residues should be described in detail. Alternatively, this sentence could be removed from the manuscript.

We agree with the reviewer’s suggestion that the chemical environment around two arginines should be described. However, the limited resolution of our structure makes further discussion of the chemical environment difficult, and further experiments, such as mutagenesis, are needed to investigate the role of these residues. Therefore, we removed the sentence from our manuscript. And, we also modified Figure 3D along with the removal.

https://doi.org/10.7554/eLife.56862.sa2

Article and author information

Author details

  1. Jun-ichi Kishikawa

    1. Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto, Japan
    2. Institute for Protein Research, Osaka University, Suita, Japan
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Atsuko Nakanishi
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3913-7330
  2. Atsuko Nakanishi

    1. Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto, Japan
    2. Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Research Center for Ultra-High Voltage Electron Microscopy, Mihogaoka, Osaka, Japan
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - original draft
    Contributed equally with
    Jun-ichi Kishikawa
    Competing interests
    No competing interests declared
  3. Aya Furuta

    Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto, Japan
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Takayuki Kato

    1. Institute for Protein Research, Osaka University, Suita, Japan
    2. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan
    Contribution
    Resources, Data curation, Supervision, Writing - original draft
    Competing interests
    No competing interests declared
  5. Keiichi Namba

    1. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan
    2. RIKEN Center for Biosystems Dynamics Research and SPring-8 Center, Suita, Japan
    3. JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Suita, Japan
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared
  6. Masatada Tamakoshi

    Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo, Japan
    Contribution
    Resources
    Competing interests
    No competing interests declared
  7. Kaoru Mitsuoka

    Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Research Center for Ultra-High Voltage Electron Microscopy, Mihogaoka, Osaka, Japan
    Contribution
    Resources, Data curation, Supervision, Funding acquisition, Writing - original draft
    Competing interests
    No competing interests declared
  8. Ken Yokoyama

    Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto, Japan
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing
    For correspondence
    yokoken@cc.kyoto-su.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6813-1096

Funding

Japan Society for the Promotion of Science (17H03648)

  • Ken Yokoyama

Japan Agency for Medical Research and Development (JP17am0101001)

  • Kaoru Mitsuoka

Ministry of Education, Culture, Sports, Science, and Technology (12024046)

  • Kaoru Mitsuoka

Takeda Science Foundation

  • Ken Yokoyama

Japan Science and Technology Agency (JPMJCR1865)

  • Kaoru Mitsuoka

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to all the members of the Yokoyama Lab for their continuous support and technical assistance. Our research was supported by Grant-in-Aid for Scientific Research (JSPS KAKENHI), Grant Number 17H03648, and Takeda Science foundation to KY Our research was also supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP17am0101001 (support number 1312), and Grants-in-Aid from ‘Nanotechnology Platform’ of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to KM (Project Number. 12024046). This work was also supported by JST CREST to K.M. (Grant Number. JPMJCR1865).

Senior Editor

  1. Olga Boudker, Weill Cornell Medicine, United States

Reviewing Editor

  1. David M Kramer, Michigan State University, United States

Reviewer

  1. Geoffry A Davis, Michigan State University, United States

Publication history

  1. Received: March 12, 2020
  2. Accepted: July 7, 2020
  3. Accepted Manuscript published: July 8, 2020 (version 1)
  4. Version of Record published: July 17, 2020 (version 2)

Copyright

© 2020, Kishikawa et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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