The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 moiety, with proton flow through the Vo membrane moiety, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo of eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo 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 the isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo adopts the auto-inhibited form and how the holo complex prevents the formation of this inhibited Vo form. One Sentence Summary Cryo-EM structures of rotary V-ATPase reveal the ON-OFF switching mechanism of H+ translocation in the Vo membrane domain.


CryoEM structures of the isolated Vo and holo Tth V/A-ATPase 1
We purified both Tth V/A-ATPase and Vo via a His3-tagged c subunit from 2 membranes of T. thermophilus cells using Ni-NTA resin. For Tth V/A-ATPase, 3 acquisition of micrographs was carried out using a Titan Krios equipped with a Falcon II 4 direct electron detector. Cryo-EM micrographs of the complexes reconstituted into 5 nanodiscs resulted in higher resolution EM maps compared with the LMNG solubilized 6 preparation previous reported (17). The strategy of single particle analysis for Tth V/A-7 ATPase is summarized in Figure S2A. The final structure of state 1 has an overall 8 resolution of 3.6 Å ( Figure 2A). After subtraction of the EM density of the membrane 9 embedded domain from the density of the whole complex, we obtained a focused density 10 map of A3B3D1F1d1 with two EG peripheral stalks and the soluble arm domain of the a 11 subunit (asol) at 3.5 Å resolution. This map allowed us to build an atomic model of 12 A3B3D1F1 (V1). In our map, the obvious density of ADP-Mg was observed in the closed 13 catalytic site, but not clearly observed in semi-closed site, in contrast to our previous 14 structure of state 1 (5Y5Y). The secondary ADP in the semi-closed site shows lower 15 occupancy, it is due to the low affinity of the semi-closed site for nucleotide and partial 16 flexibility in the complex ( Figure S3A). In the recent cryoEM map of Tth V/A-ATPase 1 (6QUM), clear densities likely to correspond to ADP were observed in the cavities of the 2 crown-like structure formed by the six β barrel domains of A3B3 (27). In contrast, these 3 densities were not clearly visible in our structure ( Figure S3B). These differences are 4 presumably due to the purification procedures; we purified the His-tagged Tth V/A-5 ATPase using a nickel column, while the authors of the other study isolated their Tth V/A-6 ATPase without affinity purification. 7 Purified Vo reconstituted into nanodiscs was subjected to single particle 8 analysis using a cryoEM (CRYOARM200, JEOL) equipped with a K2 summit electron 9 direct detector in electron counting mode. The 2D class averages showed the isolated 10 Vo with clearly visible transmembrane helices and a hydrophilic domain extending above 11 the integral membrane region ( Figure S2C). The density for the scaffold proteins and 12 lipids of the nanodiscs is clearly visible surrounding the membrane domain of the isolated 13 Vo. Following 3D classification of the Vo, only one major class was identified indicating 14 that the isolated Vo is very structurally homogenous, in contrast to the Tth V/A-ATPase 15 which is clearly visible in three different rotational states (17). Our 3D reconstruction 16 map of the isolated Vo was obtained with an overall resolution of 3.9 Å. The final map 1 shows clear density for protein components of Vo, including subunit a, d, c12 ring, but the 2 EM density for both EG stalks, which attach to the asol, is weak indicating disorder in 3 these regions, suggesting their flexibility ( Figure 2B). In this structure, the C-terminal 4 region of the EG stalk on the distal side is visible. With the exception of these two EG 5 stalks, side-chain densities were visible for most of the proteins in the complex, allowing 6 construction of a de novo atomic model using phenix and coot ( Figure 3A,B).
The map 7 contains an apparent density inside the c12 rotor ring, likely corresponding to the 8 phospholipids capping the hole of the ring ( Figure S4A). A further apparent density was 9 identified in the cavity between the a subunit and the c12 ring on the upper periplasmic 10 side ( Figure S4B). This also might be corresponded to phospholipid and we postulate 11 that this functions to plug the cavity between the a subunit and the c12 ring preventing 12 proton leak from the periplasmic proton pathway. The densities corresponding to these 13 phospholipids in our Vo structure are also observed in recently published cryoEM density 14 map of the holo complex (27). Notably, the diameter of the c12 rotor ring in the isolated 15 Vo is slightly smaller than that in the Tth V/A-ATPase ( Figure S5A). It is likely that 16 penetration of the short helix of the subunit D into the cavity of subunit d enlarges the 1 diameter of the c12 rotor ring in the Tth V/A-ATPase. 2

Structure comparison of the isolated Vo with the holo complex 3
A comparison of our structure of the isolated Vo with that of Vo moiety in holo 4 complex revealed a high degree of similarity in the membrane embedded region. 5 However, there were significant differences in the a subunit. The basic structure of the 6 a subunit of Tth Vo is almost identical to the eukaryotic counterpart, with both composed 7 of a soluble arm domain (asol) and a C-terminal hydrophobic domain responsible for 8 proton translocation via rotation of the c12 ring. The asol contains two globular α/β 9 folding subdomains responsible for binding of both the proximal and distal EG stalks 10 ( Figure 3A and B). Both globular subdomains are connected by a hydrophilic coiled 11 coil with a bent conformation. 12 In contrast to the structure of Vo moiety in the holo complex, the asol in Vo only 13 is in close proximity to the d subunit as a result of kinking and twisting of the coiled coil 14 at residues a/L119 and a/A246 ( Figure 3C, indicated by the arrows). In this structure, 15 there are several interactions between the residues in the asol and the d subunit ( Figure  16 3D). At the proximal site, three amino acid residues, a/E57, a /H65, and a/Q106, form 1 salt bridges or hydrogen bonds with residues d/R38, d/S41, and d/R64 in the d subunit, 2 respectively. The side chain of d/R59 likely forms π-π stacking with a/R103. Our 3 structure also revealed clear connected densities between the distal subdomain of the asol 4 and the d subunit ( Figure 3E Voltage threshold for proton conductance activity of the isolated Vo 12 Our structure of the isolated Vo suggests that the rotation of c12 rotor ring 13 relative to the stator is mechanically hindered by a defined interaction between the asol 14 and d subunit. To investigate this mechanical hindrance of proton conductance through 15 the Vo, we reconstituted the isolated Vo into liposomes energized with a Δψ generated 16 through a potassium ion (K + )/valinomycine diffusion potential. The pH change in the 1 liposomes was monitored with 9-Amino-6-Chloro-2-Methoxyacridine (ACMA); the 2 emission traces at 510 nm excited at 460 nm were recorded (Figure 4). The size of the 3 membrane potential was modulated by varying the external K + concentration. As shown 4 in Figure 4B, a voltage threshold was observed in that the isolated Vo shows no proton 5 conductance at less than 120 mV of membrane potential. When the membrane potential 6 is 130 mV or more, the proton conductance through the Vo increases in proportion to the 7 membrane potential ( Figure 4B). The reported membrane potential in bacteria cells is -8 75 ~ -140 mV (28). Thus, the observed inhibitory mechanism of the isolated Vo can 9 function to prevent proton leak through the Vo under physiological conditions. In 10 contrast to the Vo, several experiments have indicated that proton conductance through 11 Fo of bacteria does not show the threshold of membrane potential (29). Together, the 12 observed results strongly suggest that the asol of the a subunit and the d subunit, absent in 13 Fo and hallmarks structure of the V type ATPases, are key for mechanical inhibition of 14 proton conductance through Vo. 15

Structure of the membrane embedded region of the isolated Vo 16
Our atomic model of Vo presented here reveals details of both proton paths formed by the 1 membrane embedded C-terminal region of the a subunit (aCT) and its interface with the 2 c12 ring. The aCT contains eight membrane embedded helices, MH1 to MH8. MH7 3 and MH8 are highly tilted membrane embedded helices characteristic of rotary ATPases. 4 The cytoplasmic hydrophilic cavity is formed by the cytoplasmic side of MH4, MH5, 5 MH7, and MH8, and the c subunit /chainZ. The cavity is lined by polar residues, a/R482, 6 a/H491, a/H494, a/E497, a/Y501, a/E550, a/Q554, a/T553, a/H557, and c(Z)/Thr54 7 ( Figure 5A), which seem to make up the cytoplasmic proton path. The periplasmic sides 8 of MH1, MH2, MH7 and MH8 form the periplasmic hydrophilic cavity, lined with 9 a/D365, a/Y368, a/E426, a/H452, a/R453, a/D455, and c(Y)/E63. The two hydrophilic 10 channels are separated by a salt bridge formed between c(Z)/63Glu, a residue critical for 11 proton translocation, and a/Arg563, a/Arg622 and a/Gln619 of MH7 ( Figure 5B). This 12 salt bridge is conserved in both eukaryotic and prokaryotic Vo (25,26). In contrast the 13 salt bridge forms between a single arginine residue and a single glutamic (or aspartic) 14 acid residue in Fo(5,30,31). Similar to the two channel model described for other rotary 15 protonation and deprotonation of the carboxy groups on the c12 ring, with the resulting 1 rotation of dc12 driven by proton translocation from periplasmic to cytoplasmic sides. 2 Notably, in addition to the rigid salt bridge formed between the two a/Arg residues, a/Gln 3 and c/Glu, interactions between the act and c12 ring are observed; a/Asp392 and Leu393 -4 c(Y)/Arg49 in the loop region of the c subunit ( Figure S6A), and the periplasmic sides of 5 MH5 and MH6 are in close proximity to the C-terminal end of the c subunit ( Figure S6B). 6 Overall, our Vo structure is largely identical to the Vo moiety in holo complex with the 7 exception of key alterations in hydrophilic domain (27). 8

Molecular basis of the auto-inhibition of proton conductance in the isolated Vo 9
The inhibition mechanism of Vo depends upon conformational changes in two 10 subunits. In the isolated Vo, the d subunit adopts the closed form in which three side 11 chains of the d subunit are able to interact with the distal subdomain of asol. Once the 12 short helix of the D subunit inserts into the cavity of the d subunit, the interaction between 13 H6 and H11 via d/R90 and d/E195 is broken ( Figure 6A and Movie S1), resulting in the 14 d subunit adopting an open form where the orientation of three side chains move away 15 from the distal subdomain of asol. 16 1 of the distal EG stalk to the top of the A3B3. In the isolated Vo, the C-terminal region of 2 the EG stalk binding onto the distal subdomain of asol is at a much steeper angle relative 3 to the horizontal coiled coil structure of asol than that in the holo enzyme ( Figure 6B, C 4 and S7). Once the N-terminal globular domain of the distal EG stalk binds onto the top 5 of A3B3, the angled distal EG adopts a vertical standing form, resulting in both a twisting 6 and kinking of the coiled coil of the hydrophilic arm and the distal globular subdomain 7 ( Figure 6C, Movie S2). These dynamic motions of the asol of a subunit induces 8 disruption of the specific interactions of asol with d subunit. 9 The isolated yeast Vo also adopts a similar inhibited conformation where the 10 asol is in close proximity to the d subunit, resulting in interaction between the stator and 11 the rotor and inhibition of proton conductance (24,25). Although an atomic model of 12 yeast holo V-ATPase has yet to be determined, the asol is some distance from the d subunit With this single exception, the eukaryotic and prokaryotic V-ATPases seem to share a 2 similar auto-inhibited mechanism of Vo preventing proton leakage from cells or acidic 3 vesicles. This suggests that the auto-inhibition mechanism of Vo is conserved during 4 the evolution of V type ATPases. 5 The interaction between the asol and d subunit stabilizes the isolated Vo structure 6 and protects against loss of d-subunit in the absence of the rotor-stator interactions 7 mediated by V1 as a result of the dissociation of the two domains (35). This stabilization 8 of Vo is most likely to be key for both assembly of holo V-type ATPase complexes and 9 regulation of eukaryotic V-ATPase via dissociation of V1 from Vo. The proximal and distal subdomains of a-subunit are circled by the dotted lines. C. 4 Comparison of the relative positions of asol (red) and the d subunit (green) in the isolated 5 Vo (left) and the Vo moiety in the holo complex (right). Arrows indicate the kinking and 6