SCIENCE ADVANCES | RESEARCH ARTICLE
BIOCHEMISTRY

Metastable asymmetrical structureof a shaftless V1 motor
Shintaro Maruyama1*, Kano Suzuki1*, Motonori Imamura2, Hikaru Sasaki1, Hideyuki Matsunami3,
Kenji Mizutani1,4,5, Yasuko Saito6, Fabiana L. Imai1, Yoshiko Ishizuka-Katsura3,
Tomomi Kimura-Someya3, Mikako Shirouzu3, Takayuki Uchihashi7,8,
Toshio Ando2,7, Ichiro Yamato1,6, Takeshi Murata1,3,4,9†

INTRODUCTION

The quaternary structure of a protein is thought to be the basisof the cooperativity of its subunits, as seen in hemoglobin and multimeric ring complexes including helicases and AAA-type adenosine triphosphatases (ATPases) (1). F-, A-, and V-ATPases are
unique rotary motors that also contain these ring structures intheir catalytic domains and are capable of pumping ions by using
adenosine 5′-triphosphate (ATP) hydrolysis energy (2). F-ATPases
work as ATP synthases in mitochondria, chloroplasts, and oxidative
bacteria (3). In Archaea, A-ATPases work as ATP synthases like
F-ATPase, although their structure and subunit composition resemble those of V-ATPases (4). V-ATPases work as proton-­transporting
ATPases in various organelles and plasma membranes of eukaryotic cells (2). These ATPases consist of hydrophilic globular catalytic
domains (F1, A1, or V1) and hydrophobic membrane-embedded
domains (Fo, Ao, or Vo), which facilitate ion translocation across membranes (2–4). The rotational catalysis mechanisms of these ATPases
have been proposed on the basis of single-molecule observations(5, 6, 7) and structural studies (8–13). However, the principle ofthe cooperative transition mechanism of the rotary motors remains
unclear.
V-ATPases are also found in bacteria (14, 15). We have been
studying the structure and function of bacterial V-ATPase fromEnterococcus hirae (15). This enzyme physiologically functions as a1

Department of Chemistry, Graduate School of Science, Chiba University, 1-33
Yayoi-cho, Inage, Chiba 263-8522, Japan. 2Nano Life Science Institute (WPI NanoLSI),
Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. 3Laboratory forProtein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics
Research, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. 4Molecular
Chirality Research, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
5
Graduate School of Medical Life Science, Yokohama City University, 1-7-29
Suehiro-­cho, Tsurumi, Yokohama 230-0045, Japan. 6Department of Biological Scienceand Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo
125-8585, Japan. 7CREST/JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
8Department of Physics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi
464-8602, Japan. 9JST, PRESTO, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
*These authors contributed equally to this work.
†Corresponding author. Email: t.murata@faculty.chiba-u.jp
Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019

primary ion (Na+) pump (16) and is composed of nine subunits,with amino acid sequences that are homologous to those of thecorresponding subunits of eukaryotic V-ATPases (15). We haveestablished the in vitro expression, purification, and reconstitution
systems of E. hirae V1-ATPase from A3B3 and DF complexes (17)and have elucidated several crystal structures (18, 19) and direct rotational dynamics of the V1 complex (20, 21) by x-ray crystallography and single-molecule observations, respectively. On the basis ofthese findings, we have proposed a rotation mechanism model ofthe V1-ATPase, in which the fundamental principle of the cooperative function is not yet defined (19).
We have also solved the crystal structures of A3B3 complex of
E. hirae V1-ATPase (Fig. 1A), which is composed of three A1B1 pairsthat form a hexamer ring. Each A1B1 pair as a unit consists of a catalytic subunit (A) and a noncatalytic subunit (B). To form the A3B3
ring, the N-terminal -barrel domain is postulated to operate like asa headband (18) to form a crown structure (Fig. 1B). The A3B3 complex, consisting of three identical A1B1 units, has an asymmetricalhexamer ring structure even in the absence of bound nucleotides(eA3B3) (18). The central domains of the A and B subunits in thehexamer are in close contact and are densely packed, probably because of the tight packing of the crown structure, which may impart
stress to the subunits that, in turn, produces a strain that forms theasymmetrical arrangement (Fig. 1C). As a result, the three A1B1
units are composed of three different conformations with different
nucleotide binding affinities: “empty” (ATP-unbound form incapable of nucleotide binding), “bindable” (ATP-accessible form), and“bound” (ATP-bound form) forms (Fig. 1D) (18). The asymmetricalstructure of nucleotide-free A3B3 complex (eA3B3) is changed upon
the binding of the nonhydrolyzable ATP analog, adenosine 5′-(,-imino) triphosphate (AMP-PNP). The binding of AMP-PNP to thebound and bindable forms triggers a conformational change of thebindable form to the bound form (bA3B3; Fig. 1E). These structures
suggest that the A3B3 complex cooperatively changes its conformation from one asymmetrical structure to another by the binding anddissociation of nucleotides in one rotational direction, which determines the order of ATP hydrolysis (18). Furthermore, the structure
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V1-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A3B3 complex and a central DF shaft.
The nucleotide-free A3B3 complex of Enterococcus hirae, composed of three identical A1B1 heterodimers, showeda unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, whichforms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A3B3. The nucleotide-freemutant A3B3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A3B3
forms a metastable (stressed) asymmetric structure composed of unstable A1B1 conformers due to the strong
constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

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License 4.0 (CC BY-NC).

SCIENCE ADVANCES | RESEARCH ARTICLE
A

N-terminal domain B

C

Central domain

C-terminal domain

t

Empty

Ti
gh

(2
)

d
un

d
un
Bo

d
un
Bo

ble

da

Bin

)

(1

Bo

F

Empty

nd

E

Empty

Bo
u

D

of bA3B3 is changed by the binding of the DF complex inside thering, which results in the formation of a more tightly packed “tight”
structure compared to the bound structure formed in A3B3 complex
with bound AMP-PNP (bV1; Fig. 1F). The tight form presumably
corresponds to the “catalytic dwell” waiting for ATP hydrolysis tooccur in the rotary cycles (18, 19).
To elucidate the mechanisms of the asymmetrical structure formation and the cooperative conformational change depending on nucleotide binding to the A3B3 complex, we designed a loosely packed
A3B3 mutant of E. hirae by weakening the crown structure. The
three-dimensional (3D) structure of the mutant was studied using
high-speed atomic force microscopy (HS-AFM) and x-ray crystallography. By comparing the structures of the mutant and wild-type
A3B3, we clarified the mechanism of the asymmetrical structure formation. Owing to the weak force exerted by the mutant crown, themutant A3B3 in the absence of bound nucleotides had a nearly symmetrical structure, with more open A1B1 units than the wild type,indicating that the crown is the key determinant of the asymmetryof the A3B3 structure. In addition, a tight form having a closely
packed A1B1 structure was observed in the mutant A3B3 in the presence of bound AMP-PNP, even without the DF shaft. On the basis ofthese findings, we propose that the origin of the cooperative activityof the subunits, as observed for V-ATPase, is from the stress-strain
within the molecular rotary motors, which work by transitioning
between metastable conformations.
RESULTS

Sample preparation and biochemical properties
To weaken the crown structure of E. hirae A3B3, we mutated anamino acid at the hydrophobic boundary of the -barrel domain
connecting the three A1B1 units by referring to the 3D structures of
the complex (18). We replaced the leucine residue at amino acid 65(Leu65) in the B subunit with tyrosine (Tyr), which is more hydrophilic and bulky. The L65Y mutant B has weaker hydrophobic interMaruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019

actions within the crown structure, which was expected to produce
a less stable ring formation of the composite units in the mutant.
We expressed the mutant B (L65Y) and unchanged A proteins in an
Escherichia coli cell-free protein expression system. The mutant B(L65Y) and unchanged A subunits were purified similar to the wildtype A3B3 complex, even with a weakened crown (fig. S1). To evaluate the subunit stoichiometry of those complexes in solution, negative
staining electron microscopy (EM) was done using a Tecnai F20
device. The examinations revealed a ring-shaped hexamer with an
A and B subunit stoichiometry of 3:3 (fig. S2).
ATPase activities of the purified wild-type A3B3 and mutant
A3B(L65Y)3 complexes were measured to compare their integrity (fig. S1).
The initial velocity of the specific activity of the mutant was approximately 40% that of the wild-type activity. The activity of the mutant
decreased rapidly, in contrast to the gradual decrease observed in thewild-type activity. Following incubation with the DF complex, theactivity of wild-type enzyme increased by approximately 2.9 times,suggesting that the V1 complex was reconstituted with the DF shaft.
The mutant A3B(L65Y)3 showed similar enhancement of activity following incubation with DF, suggesting that the mutation had notaffected the interaction with the shaft, thus maintaining its integrity.
However, the mutant activity was lower than that of the wild type,possibly because the weakened crown caused a gradual loss of complex stability. These results indicate that the mutant A3B(L65Y)3 retains the ability to interact with DF shaft but is less stable.
HS-AFM of wild-type and mutant A3B3
To compare the structural differences between the wild-type A3B3
and mutant A3B(L65Y)3 complexes in the absence of nucleotides, we used
HS-AFM, which is a unique tool capable of visualizing the structureand dynamics of proteins, nucleic acids, cells, and organelles (22). HSAFM can capture images of individual biomolecules in aqueous solution at a lateral resolution of approximately 2 nm, a vertical resolutionof approximately 0.1 nm, and a time resolution of approximately
100 ms (22, 23). It can observe a single molecule for many frames in
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Fig. 1. Crystal structures of the A3B3 and A3B3DF (V1) complexes. (A) Side view of the nucleotide-free A3B3 (eA3B3) structure divided into three domains as shown with
two lines: N-terminal [residues A (1–71) and B (1–77)], central [residues A (72–449) and B (78–362)], and C-terminal [residues A (450–593) and B (363–458)] domains. A and
B subunits are depicted in blue and purple, respectively. (B to F) Top views of N-terminal -barrel domain (crown) (B), central domain (C), and C-terminal domains of eA3B3(D), AMP-PNP bound A3B3 (bA3B3) (E), and AMP-PNP bound V1 (bV1) (F) as surface representations viewed from the N-terminal -barrel side. Red arrows indicate the nucleotide binding sites. The bound AMP-PNP molecules are shown as red spheres. The DF shaft is shown in green.

SCIENCE ADVANCES | RESEARCH ARTICLE

A

Wild-type A3B3
#1
D

Tip

#2

is larger than the B subunit by approximately 140 amino acids)(Fig. 2A), HS-AFM examination of the A3B3 complex revealed thehexamer ring structure with three higher (bright) spots corresponding to the A subunits for both the wild-type and weak mutant (Fig. 2,
B and C). The height difference between the A and B subunits
was 0.1 to 0.5 nm for the wild-type and 0.55 nm for the mutant. To
eliminate the noise derived from HS-AFM and positional fluctuations of the molecules, we averaged successive HS-AFM images
for each molecule. In addition, by enhancing the image contrast,
we were able to visualize each subunit of the A3B3 hexamer (Fig. 2,
D and F, and movie S1). Enhancing the contrast further revealed
that one of the three A subunits was lower (darker) in the wild-type
A3B3 (Fig. 2E). All the images used for averaging were aligned withthe same orientation of the A subunit height. The difference in thelowest and highest height of the A subunits was 0.3 ± 0.1 nm. Considering the vertical resolution of HS-AFM, this difference in size
was significant and allowed us to conclude that the wild-type A3B3
complex took a similar asymmetric form in aqueous solution and in
the crystals (Fig. 1D). By contrast, the mutant A3B(L65Y)3 did notshow a clear difference in height among the three A subunits(Fig. 2G), suggesting that the L65Y mutation in the B subunits
makes the A3B(L65Y)3 structure more symmetrical, as expectedfrom the reduced interaction between the A and B subunits in the
mutant complex.

#3

#4

#5
0 nm

B
A
−1.5 nm

B

5 nm

E

Wild-type A3B3

0 nm

8 nm

−0.5 nm
0 nm
7.5 nm
10 nm
0

C

10

20
nm

30

Mutant A3B(L65Y)3
#1
F

#2

#3

#4

#5
0 nm

Mutant A3B(L65Y)3
8 nm−1.5 nm

G

5 nm
0 nm

0 nm
7.5 nm−0.5 nm

10 nm

0

10

20
nm

30

Fig. 2. HS-AFM images of the wild-type and mutant A3B3 complexes. (A) Schematic image of HS-AFM imaging observation of A3B3 from the side with the N-terminal domain at the bottom. A and B subunits are shown in blue and purple, respectively, as a surface representation. The tip of the short cantilever is also drawn. (B and C) HS-AFM
images of wild-type A3B3 (B) and mutant A3B(L65Y)3 (C) showing 50 nm × 50 nm area. The insets (30 nm × 8 nm) are the height line profiles from the path indicated by awhite line in the images with the height of the highest peak marked. (D and E) Five representative HS-AFM images of wild-type A3B3 as averaged images, with the contrastof the height color table adjusted (see Materials and Methods). An A subunit (higher subunit) and a B subunit (lower subunit) are indicated at #1 in (D). The orientations of
averaged images were adjusted to be the same for all molecules. Scale bar is indicated by a black bar and denotes 5 nm. (F and G) Five representative averaged images ofthe mutant A3B(L65Y)3. Zero nanometer of the color maps in (B) and (C) is the lowest point of the images, whereas in (D) to (G) it is the highest.
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Downloaded from http://advances.sciencemag.org/ on March 8, 2019solution. Therefore, HS-AFM is a useful tool to understand the structure of individual molecules, thus providing a different perspective from other techniques such as crystallography, which averages
structures from thousands of different molecules. In addition, HSAFM is very sensitive to height (approximately 1 Å within the same
AFM image), in some cases providing a higher resolution than acryo-EM. For these reasons, evaluating the structural differences using
HS-AFM is a reasonable choice. Previous HS-AFM observations ofthe 33 hexamer ring of F1-ATPase, which has a shape resembling
the A3B3 hexamer ring of V1-ATPase, showed changes of the 33
complex from symmetrical to asymmetrical ring shape upon the
addition of ATP (7) [unlike the A3B3 complex, the 33 complex has
a symmetrical structure in the absence of nucleotides (24)]. Therefore,
it seemed likely that HS-AFM would also detect the structural differences between the wild-type A3B3 and the mutant A3B(L65Y)3.
The A3B3 complex was deposited on the mica surface using thesame condition as 33 hexamer ring of F1-ATPase, where the mica
surface was chemically treated with 3-aminopropyl-triethoxysilane(APTES) followed by glutaraldehyde fixation to attach the N-terminalside of the A3B3 complex to the surface (7). This permitted the examination of the C-terminal side of the A3B3 complex, which is theregion where the complex interacts with the central DF complex.
Consistent with the crystal structure of the A3B3 complex and the
known size difference between the A and B subunits (the A subunit

SCIENCE ADVANCES | RESEARCH ARTICLE

A

B

sence of nucleotides (Fig. 1D) (18). All three A1B(L65Y)1 units in the
mutant complex were found to be most similar to the bindable form
among the possible A1B1 unit forms (empty, bindable, bound, andtight) by comparing the RMSD values (table S2), contact surface
areas (table S3), and structures of the substrate binding sites (fig. S4).
From the previous (18, 19) and present findings, we propose thatthe bindable form is a preferable default structure for the A1B1 unit
when connected with the -barrel domain in the absence of boundnucleotides. Thus, the weak crown of the mutant relaxed the dense
packing stress of the central domain (Fig. 3B and table S4) by maintaining similarly close interunit contact surface area (approximately
2700 Å2), but with marginally smaller intraunit area (4547.3 Å2) thanthat of the wild-type (4837.0 Å2), and finally expanded the complex
to allow more open symmetrical C-terminal halves in all bindable
forms (Fig. 3C and movie S3).
Crystal structure of AMP-PNP bound mutant A3B(L65Y)3
The crystal structure of the wild-type bA3B3 in the presence of excess AMP-PNP revealed a hexameric ring structure similar to thatof eA3B3, and two of the three nucleotide binding sites were occupied with AMP-PNP (Fig. 1E). We crystallized the mutant A3B(L65Y)3
complex [bA3B(L65Y)3] in the presence of excess AMP-PNP and determined the crystal structure at a resolution of 2.1 Å (table S1 andFig. 4A; PDB ID: 5ZE9). The crystal structure revealed a hexamer
similar to that of bA3B3; however, one of the three mutated sites in
the crown was disrupted. As a consequence, the three A1B(L65Y)1 units
formed a spiral-like hexameric structure (Fig. 4, A to D, and movie S4).
The nucleotide binding sites of A1B(L65Y)1 units were all occupiedC

Bindable(1)

nd

Bi
ab
le (2

Bi

)

D

E

e (3)

bl

a
nd

F

E261
R262

K238

T239

Fig. 3. Crystal structure of nucleotide-free A3B(L65Y)3. (A) Top view of N-terminal -barrel (or crown) structure shown as in Fig. 1B. The mutated residues are shown inyellow. (B) Top view of the central domain of eA3B(L65Y)3 shown as in Fig. 1C. (C) Top view of the C-terminal domain of eA3B(L65Y)3 as in Fig. 1D, which is superimposed at
the N-terminal -barrel domain of the bindable form (chains B and E) of A1B(L65Y)1 units onto that of eA3B3 (gray). (D and E) Superimposed structures using C atoms of
three A (blue) and B (purple) subunits in eA3B(L65Y)3. The P-loop and arm are shown in yellow and white, respectively. The green box indicates the nucleotide binding site.
(F) Magnified views of the nucleotide binding sites with conserved residues, corresponding to the green box in (E).
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Crystal structure of nucleotide-free mutant A3B(L65Y)3
We crystallized the mutant A3B(L65Y)3 complex to elucidate detailed
structural differences from wild-type eA3B3 and obtained a crystal
structure in the absence of nucleotides at 3.4 Å resolution [Protein
Data Bank (PDB) ID: 5ZEA; table S1, denoted as eA3B(L65Y)3]. In the
crystal lattice, three of the A1B(L65Y)1 units formed a hexagonal arrangement similar to that of the wild-type eA3B3 (Fig. 3, A and B),
which indicated the tendency of the A1B(L65Y)1 overall geometricalstructure to form a ring, as in A3B(L65Y)3. Two hexamer A3B(L65Y)3
molecules, which were designated eA3B(L65Y)3-1 (chains A to F) andeA3B(L65Y)3-2 (chains G to L), were contained in the asymmetric unitof the crystal lattice. These two structures closely resembled eachother [root mean square deviation (RMSD) = 1.30 Å]. Therefore,
we used the data from the eA3B(L65Y)3-1 (chains A to F) in all of the
figures and in the subsequent discussion.
The mutated amino acid Leu65 site had an electron density corresponding to a Tyr residue (fig. S3). Comparison of the crownstructures of wild-type and mutant A3B3 complexes revealed several
differences in the side-chain orientations around the mutated site,which suggested the loss of hydrophobic interaction (movie S2). Thismutation seemed to loosen the crown structure, with its C-terminal
halves appearing to be more open, resulting in a slightly looser
shape with weaker associations (Fig. 3C and movie S3). The three
individual A and B subunit structures in the mutant were very similar (Fig. 3, D to F). Consequently, the mutant A3B3 had a nearly
symmetrical open structure as observed in the HS-AFM images(Fig. 2, C, F, and G), while the wild-type eA3B3 displayed the asymmetrical C-terminal halves (empty, bindable, and bound) in the ab-

SCIENCE ADVANCES | RESEARCH ARTICLE
A

B

C

D

Tight(1)

nd

u
Bo
90°

E

E261
Mg2+

Mg2+

T239

R262
B/R350

K238

Bound

F

R262

AMP-PNP

90°

Mg2+

E261
T239

Mg2+

R262
B/R350

K238

Tight(1)G

AMP-PNP

90°

E261
R262

Mg2+

Mg2+

T239

R262
B/R350
K238

Tight(2)

AMP-PNP

Fig. 4. Crystal structure of AMP-PNP bound A3B(L65Y)3. (A) Side-top view of AMPPNP bound mutant A3B(L65Y)3 [bA3B(L65Y)3]. N-terminal domain is shown in surface
representation, and the central and C-terminal domains are in cartoon representations. Colors are the same as in Fig. 1E. (B and C) Top views of the N-terminal (B) andcentral (C) domains as in Fig. 3, A and B, respectively. (D) Top view of the C-terminal
domain of bA3B(L65Y)3 as in Fig. 1E. (E to G) Magnified views of the nucleotide bindingsites. The sites corresponding to the green box in Fig. 3E are shown for bA3B(L65Y)3.
The P-loop and arm are shown in yellow and white, respectively. The right panels
show the A-B interface rotated 90° around a vertical axis from the left panels, withconserved residues of the “bound” (E), “tight(1)” (F), and “tight(2)” (G) forms in bA3B(L65Y)3.
To compare structural differences, two dotted vertical lines were drawn from the Cposition of R350 in bound form (E) and tight forms (F and G) to show the closer location (approximately 1.2 Å) of the tight form to the bound nucleotides. The |Fo| − |Fc|
map calculated without AMP-PNP: Mg2+ at the binding pockets at 4.0 are shownin red (negative) and green (positive) meshes.
Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019DISCUSSION

The assembly of identical units is generally expected to form a symmetrical structure. However, the A3B3 complex in the absence ofbound nucleotides shows an asymmetrical hexagonal arrangement,
which involves the formation of a crown of the -barrel domain (18).
We confirmed the asymmetrical hexagonal arrangement of the wild-­
type A3B3 by HS-AFM imaging, which contrasted with the more
symmetrical arrangement of the mutant A3B(L65Y)3. X-ray crystallography of the mutant eA3B(L65Y)3 revealed a nearly open symmetrical structure with all bindable forms in the absence of boundnucleotides. We suggest that the bindable form is the preferred
structure of the A1B1 unit in the nucleotide-free A3B3 complex. Furthermore, the A3B(L65Y)3 mutant with bound AMP-PNP displayed aclosely packed spiral-like hexameric structure with a disrupted crown.
We further suggest that the tight form is the stable structure of theA1B1 unit in the AMP-PNP bound A3B3 complex.
On the basis of these findings, we can propose how V-ATPase
A3B3 forms an asymmetrical structure in the absence of bound nucleotides. The mutated weak crown is weak enough to relax thestress imposed by the close packing at the central domains of the
A and B (L65Y) subunits. Therefore, all three A1B(L65aY)1 units takethe preferable default open structure as the bindable form (Fig. 5A).
The wild-type crown is strong enough to constrain the structure andform the stringent ring size and shape of the wild-type A3B3. Thus,
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R262

t (2)

gh

Ti

with AMP-PNP and magnesium ion (Mg2+), as shown by the electron density maps (Fig. 4, E to G). The electron densities for the-phosphate of the AMP-PNP molecules in the two A1B(L65Y)1 units
were rather weak (Fig. 4, F and G), probably because AMP-PNP waspartially hydrolyzed. Thus, both the native AMP-PNP and the hydrolyzed product, AMP-PN, coexisted in the same crystal, because AMPPNP tended to be hydrolyzed to AMP-PN and phosphate under the
weakly acidic crystallization condition of pH 6.0 (25).
The bound nucleotide molecules interacted with Lys238 of theP-loop, Arg262 of the “arm” region in the A subunit, and Arg350 (Argfinger) in the B subunit of the three A1B(L65Y)1 units of the mutant(Fig. 4, E to G). One of the three A1B(L65Y)1 units displayed a slightly
different interaction between its Arg350 and the nucleotide from theother two units and more closely resembled the bound form observed
in bA3B3 (Fig. 4E and fig. S5). This difference in interaction was also
supported by the comparison of the contact surface areas (table S3)and RMSD values (table S5). The other two units had Arg350 C
located approximately 1.2 Å closer to the bound nucleotide than thebound form (Fig. 4, E to G). The A and B subunits of these units
were closely packed (table S3), corresponding to that of a tight form
detected only in the A1B1 unit interacting with the DF complex in
the V1 complex (18). Comparisons of the RMSD values and nucleotide binding sites also supported the assignment of the tight form
(table S5 and fig. S5). From these findings, we propose that the tightform is a stable default structure for the AMP-PNP bound A1B1
unit. The packing force of the tight form is so strong (table S3) thatthe weak crown of the mutant cannot withstand the stress of theconstricting structure caused by a hexamer ring with three boundnucleotides. Thus, the crown breaks to release the stress, which results in the formation of the spiral-like structure (Fig. 4A). A boundform in the bA3B(L65Y)3 would have been produced by the additional
stress of stacking on the opposite side of the broken pairs; the boundform is probably less stable than the tight form.

SCIENCE ADVANCES | RESEARCH ARTICLE
A

B

eA3B(L65Y)3
A

eA3B3

Weak crown

B

A

Bindable

B

Preferable

Preferable

C

Bound

Bindable

Empty

Preferable

Less
stablePreferable

Less
stableAMP-PNP binding
D

B

A

B

B

bA3B3
A

Strong crown
B

A

B

A

B

AMPP

NP

AMPPNPAMPPNPAMPPNPAMPPNP

Tight

Bound

Tight

Bound

Bound

Empty

StableLessstableStableLessstableLessstableLessstableFig. 5. Schematic structure models of the wild-type and mutant A3B3. The A3B3 complexes are pictured as a side view from the inside, with the ring cut and laid flat sothat all the subunits can be viewed simultaneously. A and B subunits are colored in blue and purple, respectively. The crown is highlighted by a light yellow box. The
dotted lines signify where the ends would connect to form the ring. Mutant (A and C) and wild-type A3B3 (B and D) models are drawn showing the conformational changes
induced by the nucleotide binding [from (A) to (C) and (B) to (D), respectively]. Yellow half-circles show the mutated amino acid, L65Y, in the crown. Corresponding to the weak
crown, mutant models show a convex crown (A) and a concave broken crown (C). AMP-PNP molecules are shown as black squares. The nucleotide binding sites are depicted
as white square boxes that are open, half-filled, or filled. The empty form has a specifically low affinity for nucleotides. Therefore, the square boxes indicating the bindingsites are distorted to look like a trapezoid. The form name is presented under each A1B1 unit with a description of the preferable (or stable) form or the less stable form.

the A3B3 complex becomes densely packed and closely contacts thecentral domains under strong stress (Fig. 5B). If the units canchange their conformations flexibly and arbitrarily, the hexamer
should remain symmetrical, even under the strong constraint of thecrown. However, the A1B1 units might have discrete structures, as
observed in eA3B3 and bA3B3. Therefore, the combination of thedifferent structures when they assemble into the A3B3 complex results in the formation of an asymmetrical structure with various
open or closed units. In this scenario, the wild-type asymmetricalstructure of A3B3 may have been produced as a metastable structurewith an open, preferable default, bindable form and with less preferable empty (ATP-unbound form with low affinity for nucleotide)and bound forms, which are forced to close by the strong constraintof the crown (Fig. 5B).
In the presence of AMP-PNP, A1B1 tends to be in a tight form.
The mutated weak crown cannot withstand the stress of forming thering shape with three tight forms, and the breakage at one site on
the crown results in the formation of a spiral-like hexamer (Fig. 5C).
The wild-type crown is strong enough to maintain the ring shapeof the A3B3 complex, and resists the stress of the closely contacting
central domains of the three tight forms. Instead, the crown forces
Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019

one A1B1 unit to release its bound nucleotide to form a weakly
packed empty form. The ring shape might be too constricted to allow the preferable open structure, bindable form, and instead mightallow only a slightly closed empty form (Fig. 5D). The other twounits can retain the bound nucleotides and adopt the bound form
instead of the tight form for the sake of size restriction and symmetry. It is functionally advantageous to attain the empty form (18) inthe presence of nucleotides. If the bindable or bound form is attainable, all the nucleotide binding sites in A3B3 might be occupied by
nucleotides at high concentrations, which could disrupt the crownstructure and the ring. The wild-type crown should be strong enoughto keep the ring constricted, as well as to form the empty form instead of the bindable form, expelling bound nucleotides and stabilizing the A3B3 ring structure. Thus, the crown appears to work tocontrol and restrict the behavior of the entire structure, as if it were
Sun Wukong’s headband in the Chinese folk tale, Journey to the West:
The headband keeps him to behave properly and considerately, preventing him from behaving freely and violently. The necessity ofthe quaternary structure of a cooperative protein like the A3B3
complex seems to produce a stress and/or strain response within
the complex.
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A

A

B

Bindableen

Brok

bA3B(L65Y)3

B

A

A

AMPPNP

B

A

B

A

BindableStrong crown

SCIENCE ADVANCES | RESEARCH ARTICLE

MATERIALS AND METHODS

Mutagenesis and preparation of proteins
To obtain weak crown mutants, we designed a mutation in thecrown structure of the A3B3 ring. On the basis of the 3D structure ofA3B3 and V1 in E. hirae (18), we selected the amino acid for mutation at the contact interfaces of three A1B1 units in the -barrel region (B-L65Y, which is not conserved in F-, A-, and V-ATPases).
Mutagenesis of the L65Y of B subunit was performed using the
QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies,
Santa Clara, CA, USA) (20).
An E. coli cell-free protein expression system was used to synthesize the A, B [or B(L65Y)], and DF complexes [D(T60C/L67C)F mutant
for single-molecule analysis (20)] using mixtures of respective plasmids harboring the corresponding genes with a modified naturalpoly-histidine (MKDHLIHNHHKHEHAHAEH) affinity tag, tobacco
etch virus cleavage site (EHLYFQG), and linker (SSGSSG) sequencesat the N terminus (17, 18). The cell-free lysate reaction mixture was
loaded onto a HisTrap HP column (GE Healthcare, Little Chalfont,Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019UK) equilibrated with buffer A [20 mM tris-HCl, 1 M NaCl, and20 mM imidazole (pH 8.5)], and bound proteins were eluted withbuffer B [20 mM tris-HCl, 500 mM NaCl, and 500 mM imidazole(pH 8.0)]. To obtain nontagged samples, the proteins were treated withtobacco etch virus protease at 4°C. The reaction solution was dialyzed overnight against buffer C [20 mM tris-HCl, 500 mM NaCl,
20 mM imidazole, and 2 mM dithiothreitol (DTT) (pH 8.5)]. The
dialysate was loaded onto a second HisTrap HP column equilibrated
with buffer A, and the flow-through fractions containing the nontagged proteins were pooled. The sample buffer was exchanged withbuffer D [20 mM tris-HCl, 20 mM NaCl, and 2 mM DTT (pH 8.5)]
using a HiPrep 26/10 desalting column (GE Healthcare), loaded onto
a HiTrap Q HP column (GE Healthcare) equilibrated with buffer D,and eluted with a linear gradient of 20 to 1000 mM NaCl.
For wild-type A3B3 and mutant A3B(L65Y)3 complexes
Eluted samples were further purified using a HiLoad 16/60 Superdex
200 pg column (GE Healthcare) equilibrated with buffer E [20 mM
tris-HCl, 10% glycerol, 100 mM NaCl, and 2 mM DTT (pH 7.5)].
Purified samples were concentrated with an Amicon Ultra-10 filter
with a molecular weight cutoff of 10,000.
For the DF complex
The eluted sample was further purified using a HiLoad 16/60 Superdex
75 pg column (GE Healthcare) equilibrated with buffer F [20 mM
tris-HCl, 150 mM NaCl, and 2 mM DTT (pH 8.0)]. Purified sample
was concentrated with an Amicon Ultra-­30 filter with a molecularweight cutoff of 3000.
EM and image processing
Purified samples were dispensed on glow-discharged carbon-coatedcopper grids. Each grid was negatively stained with 2.0% (w/v) uranyl
acetate. Micrographs were collected on a direct electron detector(FEI Falcon II) using FEI Tecnai G2 F20 (FEI, Hillsboro, OR, USA)
operating at 200 kV. For reference-free 2D class averaging, particle
images were boxed out with e2boxer.py in the EMAN2 package (29)and then processed with RELION software (30).
Measurement of ATPase activity
A3B3 or A3B(L65Y)3 (10 mg/ml protein) was reconstituted with the
DF complex (5 mg/ml protein) in buffer G [100 mM MES, 5 mM
MgSO4, 100 mM NaCl, and 10% glycerol (pH 6.0)] by incubating the
mixture on ice for 1 hour (18, 19). The ATPase activity of A3B3 or thereconstituted A3B3DF was measured using an ATP-regenerating system(18, 19). The reaction mixture contained 1 mM ATP/MgSO4, 2.5 mM
phosphoenolpyruvate, pyruvate kinase (50 g/ml), lactate dehydrogenase (50 g/ml), 0.2 mM -NADH (nicotinamide adenine dinucleotide) (dipotassium salt), and 2 to 20 g of protein in 1 ml ofreaction mixture (buffer G). The reaction was initiated by adding
ATP/MgSO4. The rate of ATP hydrolysis was monitored at 23°C bymeasuring the rate of oxidation of NADH, as determined by a decreasein absorbance at 340 nm.
Fabrication of electron beam deposit tip on the cantileverfor HS-AFM imaging
Electron beam deposit tip was grown on the original tip of a shortcantilever (model BL-AC7DS, Olympus, Tokyo, Japan) by focusing
the electron beam in an atmosphere of sublimated ferrocene gas using a field-emission scanning electron microscope (model SUPRA
40VP, Carl Zeiss, Jena, Germany) with the following conditions:working distance, 4 nm; beam aperture, 10 m; deposition time,
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The crystal structures of the shaftless motors of V/A-ATP synthase [Thermus thermophilus A3B3 complex, (26)] and F-ATP synthase [Bacillus PS3 33 complex, (24)], on the contrary, showed exact
threefold symmetry related by a crystallographic threefold axis in
those structures. There are two possibilities regarding this issue.
The electron density map in which any difference in the conformation of the complex had been averaged was not suitable for detecting subtle differences between the AB or  pairs. Thus, further
trials to determine the electron density maps of their whole structures are necessary to settle the symmetry or asymmetry issue. Another possibility is that the asymmetry of the shaftless motor of
E. hirae V-ATPase could be just an exceptional observation amongrotary motors, but the asymmetry of the E. hirae shaftless motor
provides a comprehensive insight for one directional rotation mechanism of the rotary motors as described above. Therefore, we believe that the stress concept proposed for the V-ATPase asymmetry
should be the underlying mechanism for any rotary motors. In thisrespect, the differences in the function of ATP-synthase/ATPase may
have a relationship to the differences in those structures. These
F- and A-ATPases function as ATP synthases physiologically withreversible ATPase activity, while V-ATPases function specificallyas ATP-hydrolyzing enzyme.
In turn, the structures with their respective shafts, not only bacterial V1/A1-ATPase (27) and F1-ATPase (28) but also eukaryotic
V1-ATPase (12) and F1-ATPase (8), showed asymmetry with threedifferent forms, similar to those found in E. hirae V 1-ATPase(18). E. hirae V1-ATPase showed empty, bound, and tight forms
irrespective of nucleotide binding (18). The two empty and boundforms seem to be unstable and stressed conformations within thecomplex as described above. Thus, the cooperative function of
V1-ATPase, and probably of other ATPases, must also be operativeby transitioning among the metastable structures produced by thestress and/or strain mechanism as proposed in this study. The rotary motors might be able to continuously perform their hydrolyzing
function by transitioning between several metastable asymmetricalstructures without ever attaining the most stable structure. This
stress and/or strain mechanism might be the fundamental principle
for any cooperative proteins with quaternary structures to achieve
their cooperative functions.

SCIENCE ADVANCES | RESEARCH ARTICLE
20 to 30 s (31). The grown tip was sharpened by etching using a radio­frequency plasma etcher (model PE-2000, South Bay Technology,
San Clemente, CA, USA) with the following conditions: gas composition, Ar and O2 in a 1:1 ratio; gas pressure, approximately 180 mtorr;
plasma power, 15 W; etching time, approximately 2 min. The resulting tip had a length of 200 to 500 nm and a tip end diameter of
5 to 10 nm.

Processing and analysis of HS-AFM images
HS-AFM image data were processed by a laboratory-made software
program written in Igor Pro (WaveMetrics, Lake Oswego, OR, USA).
The number of pixels of each HS-AFM image was increased twice
by linear interpolation. The molecule of interest was tracked to compensate for drift of sample position. The tracking was conducted by
calculating a coefficient of correlation between a template image
and each frame image under various lateral shifts of the latter image. The lateral shift providing the maximum coefficient value wasadopted. Successive HS-AFM images were stacked and averaged,
where frames with unclear images were removed.
Crystallization conditions
All crystallization trials were performed using the sitting-drop
vapor diffusion method at 296 K. The crystals were soaked in cryoprotectant by incrementally increasing the glycerol concentration
up to 20%. The crystals were then mounted on cryo-loops (Hampton
Research Corp., Aliso Viejo, CA, USA), flash-cooled, and stored inliquid nitrogen.
Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019

Structure determination
All x-ray diffraction data were collected from a single crystal ata cryogenic temperature (100 K). For eA3B(L65Y)3, x-ray diffraction
data were collected on beamline BL-17A ( = 0.98 Å) at the Photon
Factory (Tsukuba, Japan). The collected data were processed to 3.4 Åby x-ray detector software (XDS) (33). The structure was solved bymolecular replacement with MOLREP (34) using the wild-type A3B3
without bound nucleotides (PDB ID: 3VR2) as a search model.
For bA3B(L65Y)3, x-ray diffraction data were collected on beamline BL-1A ( = 1.1 Å) at the Photon Factory. The collected data
were processed to 2.1 Å by XDS (33). The structure was solved bymolecular replacement with MOLREP (34) using the E. hirae V1 tightform without bound nucleotide (PDB ID: 3VR4; chains C to F) as asearch model.
The atomic models were manually built using Coot (35) and iteratively refined using REFMAC5 (36) and PHENIX (37). The refinedstructures were validated with RAMPAGE (38). The crystallographic
and refinement statistics are summarized in table S1. All RMSD values
were calculated using C atoms. The RMSD values for the superimpositions for each A1B1 unit in the crystal structures are listed intables S2 and S5. PDBePISA was used for contact surface area calculation (39). Figures were prepared using PyMOL (The PyMOL Molecular Graphics System, version 1.0, Schrödinger LLC, New York, NY,
USA). The video was generated by morphing the x-ray crystal structures of nucleotide-free wild-type (eA3B3) and mutant [eA3B(L65Y)3]
A3B3 using Chimera [UCSF Chimera, an Extensible Molecular Modeling System, version 1.12.0, University of California, San Francisco(UCSF) Resource for Biocomputing, Visualization, and Informatics](40) and PyMOL.
Other methods
Protein concentrations were determined with the BCA Protein
Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA and Thermo
Fisher Scientific, Waltham, MA, USA), with bovine serum albumin
used as the standard. SDS–polyacrylamide gel electrophoresis was
carried out according to Laemmli, and gels were stained with
Coomassie Brilliant Blue. All other chemicals were obtained from
Sigma-­Aldrich Japan KK (Tokyo, Japan) or Wako Pure Chemicals
Industry Ltd. (Osaka, Japan). For crystallization screening, chemicals were obtained from Hampton Research Corp.
SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/5/1/eaau8149/DC1
Fig. S1. Purification and ATPase activity of wild-type A3B3, mutant A3B(L65Y)3, and DF complexes.

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High-speed atomic force microscopy
HS-AFM observation was performed in the tapping mode using alaboratory-made HS-AFM apparatus (23) equipped with a dynamic
feedback controller, in which the feedback gain was automaticallytuned during imaging (32). The A3B3 proteins were immobilized ona mica surface that had been coated with APTES and then treatedwith glutaraldehyde because the protein tends to move on the mica,
and hence, it is difficult to obtain enough good data for analysis. In
the previous HS-AFM publication of 33 of F1-ATPase (7), the immobilization condition was the same as in this experiment. In thatcondition, the cooperative unidirectional rotation was observed in
the presence of ATP. Therefore, we consider that the immobilization of 33 does not affect the protein structure so much (if the symmetry changes due to the immobilization, the protein cannot function
properly). Because A3B3 of V1-ATPase is a similar protein, we makethe same assumption. The mica substrate was freshly cleaved immediately before use. APTES was diluted 1:100,000 in Milli-Q water, deposited on the mica surface, and incubated for approximately 3 min.
The APTES-coated substrate was thoroughly washed with Milli-Q
water. Glutaraldehyde diluted to 0.025% was deposited on the APTES-­
treated mica and incubated for 3 min. The surface was rinsed withan observation buffer [20 mM tris-HCl (pH 7.5), 100 mM NaCl,
2 mM DTT, and 10% glycerol]. A drop (approximately 5 l) of theA3B3 protein solution diluted to concentrations of 100 to 300 nM
was loaded onto the substrate surface for 5 min. The sample wasrinsed with the observation buffer and examined. The HS-AFM
imaging conditions were as follows: cantilever free oscillation amplitude, 0.8 to 2 nmp-p; set-point amplitude, 80 to 90% of the freeoscillation amplitude; frame rate, 3 to 5 frames/s; scan and pixel
sizes, 50 nm × 50 nm with 150 × 150 pixels or 50 nm × 40 nm with150 × 120 pixels.

For A3B(L65Y)3 without nucleotides, crystals of nucleotide-free
A3B3 [eA3B(L65Y)3] [10 mg/ml in 20 mM tris-HCl (pH 7.5), 100 mM
NaCl, and 10% (v/v) glycerol] were grown at 23°C by vapor diffusion after an equal volume (0.3 l) was mixed with 0.1 M NaCl, 0.1 M
ammonium acetate, 0.1 M bis-tris propane (pH 6.5), and 28% (w/v)
PEG-3350 (polyethylene glycol, molecular weight 3350).
For A3B(L65Y)3 with AMP-PNP, crystals of AMP-PNP bound
A3B3 [bA3B(L65Y)3] [12.1 mg/ml in 20 mM tris-HCl (pH 7.5), 100 mM
NaCl, 10%(v/v) glycerol incubated with addition of 120 mM MES(pH 6.0), 6 mM AMP-PNP, and 6 mM MgCl2 for 1 hour on ice]were grown at 23°C by vapor diffusion after an equal volume(0.5 l) was mixed with 0.2 M NaCl, 0.1 M MES (pH 5.5), and 27%(w/v) PEG-3350.

SCIENCE ADVANCES | RESEARCH ARTICLE
Fig. S2. Electron micrographs of negatively stained wild-type and mutant A3B3 complexes.
Fig. S3. Comparison of the crown structures of wild-type and mutant A3B3 complexes.
Fig. S4. Magnified views of the nucleotide binding sites of the bindable forms in eA3B(L65Y)3.
Fig. S5. Comparison of the nucleotide binding sites of wild-type and mutant complexes.
Table S1. Data collection and refinement statistics.
Table S2. RMSD values in superimpositions for each A1B1 unit of eA3B(L65Y)3 in the crystal
structures of A3B3 and V1 complexes.
Table S3. Contact surface areas between A and B subunits in various A1B1 forms (Å2).
Table S4. Sum of contact surface areas at the central domain in A3B3 (Å2).
Table S5. RMSD values in superimpositions for each A1B1 unit of bA3B(L65Y)3 in the crystal
structures of A3B3 and V1 complexes.
Movie S1. AFM scan movie of the C-terminal side of the wild-type and mutant A3B3.
Movie S2. Conformational changes at the -barrel domain induced by the mutation.
Movie S3. Conformational changes at the C-terminal domain of eA3B3 induced by themutation.
Movie S4. Conformational changes of eA3B(L65Y)3 induced by binding AMP-PNP molecules.

REFERENCES AND NOTES

Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 2019

Acknowledgments: The synchrotron radiation experiments were performed at Photon
Factory (proposals 2014G-171, 2014R-51, and 2016G048). We would like to thank the beamline
staff at BL-1A and BL-17A of the Photon Factory (Tsukuba, Japan) for help during datacollection. We would also like to thank A. Imamura who assisted with English editing,
K. Hanada for technical support with plasmid preparations, and N. Kodera who gave us advice
regarding the HS-AFM observation and analysis. Molecular graphics and analyses were
performed with the UCSF Chimera package. Chimera is developed by the Resource for
Biocomputing, Visualization, and Informatics at the University of California, San Francisco(supported by NIGMS P41-GM103311). Funding: This work was supported in part by
Grants-in-aid for Scientific Research (26291009, 17H03638, and 18H05425 to T.M.; 16H00830,
16H00758, and 15H03540 to T.U.; and 26119003 and 24227005 to T.A.) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan (MEXT); by Platform Project for
Supporting Drug Discovery and Life Science Research [Basis for Supporting Innovative Drug
Discovery and Life Science Research (BINDS)] from Japan Agency for Medical Research andDevelopment (AMED) under grant nos. JP18am0101082 and JP18am0101083; and by CREST
Program (JPMJCR13M1 to T.A.) from the Japan Science and Technology Agency (JST). Author

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SCIENCE ADVANCES | RESEARCH ARTICLE
contributions: T.M. designed the study. Y.S. designed and obtained the mutant. Y.S., Y.I.-K.,
T.K.-S., and M.S. prepared the proteins. S.M. and H.S. crystallized the proteins. S.M., H.S.,
and K.M. collected x-ray data. S.M., K.S., and H.S. processed and refined x-ray data. H.M. and
M.S. performed EM analysis. M.I., T.U., and T.A. performed HS-AFM imaging. S.M., K.S., and F.L.I.
performed functional analysis. S.M., M.I., H.S., I.Y., and T.M. analyzed the results. S.M., M.I., and
H.M. prepared figures. S.M., M.I., I.Y., and T.M. wrote the paper. All authors discussed the results
and commented on the manuscript. Competing interests: The authors declare that they haveno competing interests. Data and materials availability: All data needed to evaluate theconclusions in the paper are present in the paper and/or the Supplementary Materials.
Additional data related to this paper may be requested from the authors. Coordinates andstructure factors are deposited in the PDB with ID: 5ZEA and 5ZE9.

Submitted 17 July 2018
Accepted 17 December 2018
Published 30 January 201910.1126/sciadv.aau8149

Citation: S. Maruyama, K. Suzuki, M. Imamura, H. Sasaki, H. Matsunami, K. Mizutani, Y. Saito,
F. L. Imai, Y. Ishizuka-Katsura, T. Kimura-Someya, M. Shirouzu, T. Uchihashi, T. Ando, I. Yamato,
T. Murata, Metastable asymmetrical structure of a shaftless V1 motor. Sci. Adv. 5, eaau8149(2019).

Downloaded from http://advances.sciencemag.org/ on March 8, 2019

Maruyama et al., Sci. Adv. 2019; 5 : eaau8149

30 January 201910 of 10

Metastable asymmetrical structure of a shaftless V1 motor
Shintaro Maruyama, Kano Suzuki, Motonori Imamura, Hikaru Sasaki, Hideyuki Matsunami, Kenji Mizutani, Yasuko Saito,
Fabiana L. Imai, Yoshiko Ishizuka-Katsura, Tomomi Kimura-Someya, Mikako Shirouzu, Takayuki Uchihashi, Toshio Ando,
Ichiro Yamato and Takeshi Murata

Sci Adv 5 (1), eaau8149.
DOI: 10.1126/sciadv.aau8149

http://advances.sciencemag.org/content/5/1/eaau8149

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