Science 13Mar2020

even with two copies of C1 (Fig. 1C, blue), the
latter possibly attributable to the loss of sub-
unit H. The enzyme preparation had an ATP
hydrolysis rate that decreased during hydro-
lysis, consistent with the complex lacking sub-
unit H ( 24 , 26 ) (fig. S2). Further, the preparation
did not demonstrate full coupling of ATPase
activity in the V 1 region to proton transport
through the VOregion: Bafilomycin, which
blocks the VOregion, only partially inhibited
ATPase activity. This lack of coupling could
be due to free V 1 complexes that remain ac-
tive because subunit H is not present or to the
detergent-solubilized V-ATPase disassembling
during ATP hydrolysis, as seen with theManduca
sextaenzyme ( 28 ). The extent of SidK inhibi-
tion of ATP hydrolysis activity could not be
quantified because only the SidK-bound form
of the enzyme was available, although previous
work has shown that SidK inhibitsS. cerevisiae
V-ATPase by ~40% ( 19 ).
Cryo–electron microscopy (cryo-EM) of the
preparation yielded three three-dimensional

(3D) maps, corresponding to ~120°-rotations

of the rotor subcomplex between states ( 25 ).

These rotational states of the enzyme had

overall resolutions of 3.9, 4.0, and 3.9 Å (figs.

S3 and S4). Focused refinement of rotational

state 1 was able to improve the resolution to

3.8 Å for the membrane region and 3.6 Å for

the catalytic region of the complex. Together,

these maps allowed the construction of an

atomic model for most of the complex, with a

few components—including parts of subunits

E1 and G2, the soluble N-terminal domain of

subunit a1, subunit C1, and several luminal

loops in the membrane region—modeled as

backbone with truncated side chains (Fig. 1D,

table S6, and fig. S5). Similar to the yeast VO

structure ( 8 , 29 ), no density was apparent for

the loop between residues 667 and 712 in sub-

unit a1. Owing to the averaging that occurs

during cryo-EM image analysis, the minor

population of complexes in the preparation

possessing subunit G1 rather than G2 could

produce a map that shows a weighted aver-

age of both isoforms. Alternatively, images of

complexes containing subunit G1 may be ex-

cluded during 2D- and 3D-image classification

if they do not average coherently with the

major population of complexes to produce high-

resolution map features. Where the map shows

high-resolution features for subunit G, it accom-

modates subunit G2 better than G1 (fig. S6),

which is consistent with most of the V-ATPase

complexes containing subunit G2. Interpolating

between the three rotational states produced a

movie (movie S1) that shows the conforma-

tional changes in the enzyme that couple ATP

hydrolysis in the V 1 region to proton pump-

ing through the VOregion. These changes

illustrate the flexibility of the enzyme, partic-

ularly in the peripheral stalks, subunit C1,

and N-terminal domain of subunit a1.

Previous high-resolution insight into the

structure of the eukaryotic V 1 region has been

limited to cryo-EM of the intact yeast V-ATPase

at ~7-Å resolution ( 19 , 25 , 29 ) and a 6.2- to 6.7-Å

resolution crystal structure of the autoinhibited

Abbaset al.,Science 367 , 1240–1246 (2020) 13 March 2020 2of7

E1

SidK

A

B2

G2

C1

d1

D

F

ATP6AP2/PRR

ATP6AP1/Ac45

c-ring

e2

f

a1CTD

a1NTD

Native MS of dissociated G at HCD 250V

+5

G1

G2

13621 Da (acetylated)

13578 Da (acetylated)

Calculated mass

not detected

MW=13578 ± 1 Da

Measured mass

2400 2800 3200 3600 m/z

2000

0

100

Relative peak intensity (%)

4000 6000 8000 10000 12000 14000 m/z

+19

+14

+16

+11+13

V 1 subunits

V 1 region

683369 ± 144 Da

727196 ± 121 Da

639513 ± 182 Da

102984 ± 35 Da

70852 ± 21 Da

56460 ± 1 Da

43811 ± 1 Da

34682 ± 13 Da

Measured mass

0.13%

0.13%

0.14%

0.03%

0.10%

0.01%

0%

0.10%

Difference

682452 Da

726264 Da

638641 Da

102953 Da

70782 Da

56462 Da

43811 Da

34646 Da

Calculated mass

A 3 B2 3 C1 1 D 1 E1 3 F 1 G2 3 SidK 3

A 3 B2 3 C1 2 D 1 E1 3 F 1 G2 3 SidK 3

A 3 B2 3 C1 0 D 1 E1 3 F 1 G2 3 SidK 3

A 1 SidK 1

Hspa8

B2

C1

SidK

Composition

+56

+57

+54

115000

100

Relative peak intensity (%)

12000

m/z

12500 13000 13500

kDa

198

98

62

49

38

28

14

6

3

a1

A

B2

C1

d1

SidK

D/E1

c

G2

G1

F

e2/(RNAseK)

H+

ATP

ADP

H+

Lumen

Ca2+

V-ATPase

VO region

clathrin

ATP

ADP

H+

H+

neurotransmitter

transporter

receptor

Synaptic

cleft

Dendrite

Docking

and

Priming

Fusion

Recycling

Loading Uncoating

Axon terminal

Cytoplasm

A

C

B D

Fig. 1. Overall structure of brain V-ATPase.(A) Cycle of synaptic vesicle loading,
docking and priming, fusion, and recycling. (B) SDS-PAGE of rat brain V-ATPase
isolated with 3×FLAG SidK1-278and gel filtration chromatography. (C)Nativemass
spectrometry (MS) of V 1 region (left) and native mass spectrometry of dissociated
subunit G (right) at a higher-energy collisional dissociation (HCD) voltage of 250 V.

The charge state for one peak per subunit is indicated. The table shows the

measured mass for each peak (± SD of fit) and the calculated mass depending on

subunit composition (table S5). The difference between calculated and measured

massesisindicated.m/z, mass/charge ratio. (D) Composite cryo-EM map (left) and

atomic model (right) of brain V-ATPase in rotational state 1. Scale bar, 25 Å.

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