High-Speed Atomic Force Microscopy Reveals Rotary Catalysis of
Rotorless F 1-ATPase
Takayuki Uchihashi, et al.
Science 333, 755 (2011);
DOI: 10.1126/science.1205510

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REPORTS
References and Notes
1. B. K. Burgess, D. J. Lowe, Chem. Rev. 96, 2983
(1996).
2. B. J. Hales, Adv. Inorg. Biochem. 8, 165 (1990).
3. H. Schindelin, C. Kisker, J. L. Schlessman, J. B. Howard,
D. C. Rees, Nature 387, 370 (1997).
4. C. C. Lee, Y. Hu, M. W. Ribbe, Proc. Natl. Acad.
Sci. U.S.A. 106, 9209 (2009).
5. A small-scale nitrogenase reaction typically features
0.15 mg VFe or MoFe protein, the catalytic component ofV or Mo nitrogenase. Such an assay condition wasestablished empirically some 30 years ago (6) and hassince been used as the conventional scale of in vitro
nitrogenase assays in the field.
6. B. K. Burgess, D. B. Jacobs, E. I. Stiefel, Biochim.
Biophys. Acta 614, 196 (1980).
7. C. C. Lee, Y. Hu, M. W. Ribbe, Science 329, 642(2010).
8. Based on the retention time on a gas chromatographcolumn, the low–molecular-weight hydrocarbon products
formed by the Vala70-substituted Mo nitrogenase variants
have been assigned as methane, ethylene, ethane,
propylene, and propane (9).
9. Z. Y. Yang, D. R. Dean, L. C. Seefeldt, J. Biol. Chem. 286,
19417 (2011).
10. C. K. Rofer-DePoorter, Chem. Rev. 81, 447(1981).
11. C. C. Lee, Y. Hu, M. W. Ribbe, Angew. Chem. Int. Ed. Engl.
50, 5545 (2011).
12. Materials and methods are available as supporting
material on Science Online.
13. Given the detection threshold of CH4 (0.0007 nmol
per nmol protein per min) and the activity of V
nitrogenase in CH4 production (0.12 nmol per nmol
protein per min), the Mo nitrogenase is at least 170-foldless active than its V counterpart in catalyzing theformation of CH4 from CO.
14. Only 0.04% of electrons can be traced in thehydrocarbon products formed in the Mo
nitrogenase–catalyzed reaction.

High-Speed Atomic ForceMicroscopy Reveals Rotary Catalysis
of Rotorless F1-ATPase
Takayuki Uchihashi,1,2,3* Ryota Iino,3,4,5* Toshio Ando,1,2,3† Hiroyuki Noji3,4,5†
F1 is an adenosine triphosphate (ATP)–driven motor in which three torque-generating b subunits
in the a3b3 stator ring sequentially undergo conformational changes upon ATP hydrolysis torotate the central shaft g unidirectionally. Although extensive experimental and theoreticalwork has been done, the structural basis of cooperative torque generation to realize theunidirectional rotation remains elusive. We used high-speed atomic force microscopy to showthat the rotorless F1 still “rotates”; in the isolated a3b3 stator ring, the three b subunits
cyclically propagate conformational states in the counterclockwise direction, similar to the rotary
shaft rotation in F1. The structural basis of unidirectionality is programmed in the stator ring.
These findings have implications for cooperative interplay between subunits in otherhexameric ATPases.
1-ATPase, a water-soluble portion of adenosine triphosphate (ATP) synthase (1), is arotary motor protein. The a3b3g subcomplex (referred to here as F1) suffices as the motor, in which the rotor g subunit rotates in thestator a3b3 ring upon ATP hydrolysis (2). The concept of the “rotary catalysis” of F1 was proposedon the basis of biochemical studies (3). It wasstrongly supported by the first crystal structureF

reported (4) and directly proven by observationsof rotating single molecules (5). In F1, the catalytic sites are located at the a-b interfaces, mainlyon the b subunits. In the crystal structure (4),three catalytic sites are in different nucleotidebound states; one binds to an ATP analog (aTP-bTP
in Fig. 1E), another binds to adenosine diphosphate (ADP) (aDP-bDP), and the third is unbound (aE-bE). Both bTP and bDP assume thewww.sciencemag.org

SCIENCEVOL 333

15. E. C. Carnahan, J. D. Protasiewicz, S. J. Lippard,
Acc. Chem. Res. 26, 90 (1993).
16. The effect of deuterium on nitrogenase-catalyzed
CO reduction is probably multifaceted. Apart fromthe inverse kinetic isotope effects (i.e., kH/kD < 1,
where kH and kD are the rate constants of H2O- andD2O-based reactions, respectively) of D2O that favor
the formation of deuterated products (18, 19), othersolvent effects of D2O—such as those affecting theprotein conformation, the interaction between proteins,
and the network of hydrogen bonds—have been
observed (20–23).
17. A. A. Khodadadi, R. R. Hudgins, P. L. Silveston,
Can. J. Chem. Eng. 74, 695 (1996).
18. K. A. Kurtz, P. F. Fitzpatrick, J. Am. Chem. Soc. 119, 1155
(1997).
19. D. G. Churchill, K. E. Janak, J. S. Wittenberg, G. Parkin,
J. Am. Chem. Soc. 125, 1403 (2003).
20. W. E. Karsten, C. J. Lai, P. F. Cook, J. Am. Chem. Soc.
117, 5914 (1995).
21. T. V. Morgan et al., Biochemistry 29, 3077 (1990).
22. M. Cassman, Arch. Biochem. Biophys. 165, 60(1974).
23. S. Y. Sheu, E. W. Schlag, H. L. Selzle, D. Y. Yang,
J. Phys. Chem. A 112, 797 (2008).
Acknowledgments: We thank D. C. Rees and N. Dalleskaof the California Institute of Technology (Pasadena,CA) for help with the GC-MS analysis. This work was
supported by Herman Frasch Foundation grant 617-HF07(M.W.R.) and NIH grant GM 67626 (M.W.R.).

Downloaded from www.sciencemag.org on August 4, 2011prevailing impact of heterometal chemistry on
the efficiency of the reaction; at the same time,the merging of the product profiles of the twonitrogenases in D2O points to a critical role ofcoupled electron/proton tunneling in the mechanism of this particular reaction. The discovery
that Mo nitrogenase is capable of using CO asa substrate, albeit at a considerably lower rate
than its V counterpart, more broadly supports
the hypothesis that CO reduction is an evolutionary relic of the function of this enzyme
family. The parallelism between the nitrogenasecatalyzed CO and N2 reduction—demonstrated
further by the ability of V nitrogenase to form
CH4, an analogous product of NH3—strengthens
the theory that the ancestral nitrogenase may
represent an evolutionary link between carbon
and nitrogen cycles on earth (7). In a practicalvein, the industrial Fischer-Tropsch process uses
H2, a costly syngas component, for CO reduction and shows a tendency toward excessive formation of CH4, a low-value product (17), whereas
the nitrogenase-based reaction uses H + for hydrocarbon formation and favors the formationof C-C bond over the cleavage of C-O bond.
These features make nitrogenase an attractive
template for cost-efficient hydrocarbon formation that is directed toward C-C coupling andcarbon-chain extension. Borrowing a trick or
two from this ancient enzyme family, perhaps
an efficient strategy could be developed in the
future for controlled fuel production from CO?
Only time will tell.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/333/6043/753/DC1
Materials and Methods
Figs. S1 to S4Table S1
References (24, 25)
12 April 2011; accepted 16 June 201110.1126/science.1206883

closed conformation, swinging the C-terminal
domain toward g, whereas bE assumes the open
conformation, swinging the domain away from g.
Because these two general conformational states
appear to push or be pushed by g, respectively, it
was proposed that interactions with g control theconformational and catalytic states of individual
b’s to sequentially generate torque (6). In fact,some biochemical studies are thought to suggestthat the a3b3 ring alone does not possess intrinsic cooperativity, and g mediates the interplay
among b’s (7–9). This view was reinforced bystudies showing that backward mechanical rotation of g with external force reverses the chemical reaction toward ATP synthesis (10, 11),
whereas forced forward rotation results in accelerated ATP binding (12).
1Department of Physics, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan. 2Bio–AFM Frontier ResearchCenter, College of Science & Engineering, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan. 3Core Research for
Evolutional Science and Technology, Japan Science and Technology Agency, Sanban-cho, Chiyoda-ku, Tokyo 102-0075,
Japan. 4Institute of Scientific and Industrial Research, Osaka
University, Ibaraki, Osaka 567-0047, Japan. 5Department ofApplied Chemistry, School of Engineering, The University ofTokyo, Bunkyo-ku, Tokyo 113-8656, Japan.

*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:hnoji@appchem.t.u-tokyo.ac.jp (H.N.); tando@staff.kanazawa-u.
ac.jp (T.A.)

5 AUGUST 2011755

Recently, however, this contention has beenchallenged by the finding that even when mostinteraction sites between b and g are abolished,
F1 retains catalytic power to rotate g unidirec9.3 nm

A

βE

B

5 nm

αE

αE

βE

βE

8.5 nm

E

αE

βE

αTP

βDP

5 nm

tachment of a probe onto the rotary shaft forvisualization (5), it does not allow direct examination of whether the intrinsic cooperativity inthe a3b3 ring is the core feature responsible forsequential torque generation. We clarified thisissue by directly imaging the ATP-driven conformational transition of b’s in the isolated a3b3
ring, using high-speed atomic force microscopy(AFM) (17, 18), a technique that can visualize
proteins at work in real time without probes(19, 20).
The a3b3 subcomplex (fig. S1) was covalently immobilized on a mica surface and observedwith high-speed AFM with a frame capture timeof 80 ms unless otherwise mentioned (Fig. 1 andfigs. S2 and S3). In the absence of nucleotide,
the a3b3 showed a pseudo-sixfold symmetricring in which three alternately arranged subunits
were elevated relative to the other three (Fig. 1A
and fig. S4). The simulated AFM image of the
C-terminus side of the ring constructed from thecrystal structure of the nucleotide-free a3b3
subcomplex (21) well reproduced the observed
image (Fig. 1C). This indicates that the N-terminusside was selectively attached to the mica, althougha smaller ring corresponding to the N-terminusside was occasionally observed (fig. S5). The
three b’s, which all assumed an open conformation in the crystal structure of a3b3, gave
three protruding peaks in the simulated image
as bright spots. When a nonhydrolyzable ATPanalog AMPPNP was added, the ring became
triangular and the central hole was obscured (Fig.
1D). Although the three a’s with lower protrusions retained the same conformation as thoseunder the nucleotide-free condition, two of three

C

αE

0 nm

D

tionally (13, 14). A few biochemical studies alsosuggest the intrinsic cooperativity of the a3b3
ring (15, 16). However, because conventional
single-molecule optical microscopy requires at-

F

βTP
αDP

0 nm

Fig. 1. (A) Averaged AFM image of C-terminal side of the a3b3 subcomplex without nucleotide (movie
S1). (B) C-terminal side of the crystal structure of the nucleotide-free a3b3 subcomplex [Protein Data
Bank (PDB) code 1SKY] (21). The a and b subunits are colored in cyan and pink, respectively. The
C-terminal DELSEED motif of b corresponding to the high protruding portions is highlighted in red.
(C) Simulated AFM image of the a3b3 subcomplex constructed from the structure in (B). (D) Averaged
AFM image of C-terminal side of the a3b3 subcomplex in 1 mM AMPPNP. (E) Atomic structure of thea3b3 subcomplex with bound nucleotides. This structure is obtained by removing g from the crystal
structure of F1 (PDB code 1BMF) (4). (F) Simulated AFM image constructed from the structure in (E).
The brightness of all AFM images in this paper represents the sample height but is not linearly set tohighlight the top surface structure (fig. S4).

B
29.12 sβ1

β230.80 s30.00 s30.24 sβ330.88 s31.36 s31.60 sCumulated angle (degrees)

28.56 s

C
6000

80

5000

40

Numbers of frames

A

4000
3000
2000
1000

0
40
20
0
40
20

0

Open

β3
β2
β1

0
0

10

20

30

40

0.92

0.94

Time (s)55 nm
nm

ClosedE
Cumulated umber of CCO shifts

D 1.0

0.96

0.98

1.00

Correlation coefficients50

C
O C +1

Correlation coefficients

Fig. 2. (A) Successive AFM images showing the conformational change ofCounterclockwise
β3
0.9
O
b’s in 2 mM ATP (movie S3). The highest pixel in each image is indicated
40
C C
1.0
by the red circle. Frame rate, 12.5 frames/s. (B) Time evolution of the cuClockwise
30
mulated angle of the highest pixel. The inset shows a trajectory, superimposed
C
β
0.9
2
C O -1on an AFM image, of the highest pixels corresponding to the high protrusions
20of open b’s (412 frames, movie S6). The center of rotation is defined by the
1.0
averaged x and y positions of the highest pixels, and the cumulated angles
10
β1
are calculated relative to the first frame. (C) Correlation coefficient histograms
0.9
calculated for each b designated in (A) (n = 220). An open b is used as
0
0
10
20
30
40
8
10
12
14
16
18
20
22
a reference for the analysis of each b. (D) Time courses of correlation
Time (s)Time (s)coefficients. The white and gray backgrounds show periods of O and C states,
respectively. Solid lines show the mean correlation coefficient for each period. (E) Time evolution of the cumulated number of counterclockwise shifts of the CCO
state. Circles, x’s, and crosses correspond to CCO, COO, and other irregular states (OOO and CCC), respectively. The increase in the cumulated number
indicates that the open b in CCO shifts counterclockwise.

756

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REPORTSREPORTS

B[ATP] = 2 µM

35
30
25
20
15
10
5
0
0

5

10

15

20

25

30

[ATP] = 3 µM

35
30
25
20
15
10
5
0
0

5

10

Time (s)DRate of ATP hydrolysis orconformational change (s-1)

Cumulated number of CCO shifts

C
35

15

20

25

30

3

4

Time (s)[ATP] = 4 µM

30
25
20
15
10
5

ATP hydrolysis rate
Open dwell fit
Closed dwell fit

4

2

0

0
0

5

10

15

20

25

30

0

1

2

ATP concentration (µM)

Time (s)Fig. 3. (A to C) Time evolutions of the cumulated number of counterclockwise CCO shifts at various
concentrations of ATP ([ATP]). At each [ATP], four representative curves for the different molecules areindicated as in Fig. 2E. (D) [ATP] dependence of the initial rates of ATP hydrolysis determined bybiochemical assay [circles; 2 mM, 1.6 T 0.9 s−1; 3 mM, 2.5 T 1.0 s−1; 4 mM, 3.3 T 1.3 s−1 (mean T SD, n =6 individual measurements at each [ATP])] and the rates of conformational change of b determined byfitting the dwell time histograms of open (squares; 2 mM, 1.5 s−1; 3 mM, 3.0 s−1; 4 mM, 3.8 s−1) andclosed (triangles; 2 mM, 1.5 s−1; 3 mM, 2.5 s−1; 4 mM, 3.2 s−1) states shown in fig. S11.
www.sciencemag.org

SCIENCEVOL 333

gation is unidirectional, rotating counterclockwisewith an efficiency of 83% (in 2 mM ATP) over
the total shift events examined (n = 371) (fig.
S10). Considering that two consecutive counterclockwise shifts occurring occasionally within theframe capture time (80 ms) would be counted asa clockwise shift, this value of efficiency is probably underestimated by ~6% (Fig. 3D, fig. S11,
and SOM).
The rate of rotational propagation of the CCO
state increased with increasing ATP concentration(Fig. 3, A to C), indicating that ATP binding is
rate-limiting, consistent with the biochemicallydetermined Michaelis constant of 12 mM for ATPhydrolysis (fig. S1). Histograms of the dwell timeof the O state followed a single exponential function, and those of the C state were well fitted withthe model that two consecutive events of ATP
binding to the other two b’s trigger the transitionof the b from the C to the O state (fig. S11). The
rate constants of the counterclockwise shift of
the CCO state were comparable with the initialrates of ATP hydrolysis measured biochemically(Fig. 3D). Thus, each ATP hydrolysis is wellcoupled with the open-to-closed transition of b.
Just before transitioning to the next CCO state, a
COO state occasionally appeared (~30% of totaltransitions) (x’s in Figs. 2E and 3, A to C). In
this event, one of the two closed b’s positioned
at the counterclockwise side opened in mostcases, implying that ADP can be released fromthe closed b before ATP binds to the open b(23, 24).
The present results prove that the stator a3b3
ring alone possesses high cooperativity for sequential power stroking among three catalyticb’s. This was also indicated by the observations
that the occasional subunit dissociation completely stopped the rotary propagation of the conformational state (fig. S12). Thus, the “g-dictator”
model (13), which proposes that only the interaction with g determines the conformationaland catalytic states of b’s (23, 24), is not valid.
On the other hand, the ATP-binding rate and theefficiency of unidirectionality of the a3b3 subcomplex are distinctly lower than those of F1(Fig. 3 and fig. S11). Thus, the interaction withg is dispensable but still important for the rapid
and precise rotary catalysis. Our findings are notinconsistent with the observations that the rates
and equilibriums of the catalytic reactions areapparently under the control of the rotary angleof g (10–12). The intrinsic interplay among b’s
would reinforce catalytic control by g; even if gtightly interacts with only one b, it still can act
on all b’s through b-b interplay.
These results also have implications for thecooperativity of other structurally related hexameric ATPases such as RecA- and AAA+-family
proteins, in which cooperativity among the separate catalytic sites is a central issue (25). These
ATPases may also have intrinsic cooperativity.
Comparative studies on these proteins should shedlight on the common operating principles of hexameric ATPases.

5 AUGUST 2011

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A

Cumulated number of CCO shifts

b in most cases. Thus, the high and outwardly
extended conformation propagates in the counterclockwise direction (Fig. 2, A and B).
For a more quantitative analysis of this cooperativity, the conformational states of the threeb’s in each image frame were determined fromtheir correlation coefficients, using respective reference images of b’s in the open state (fig. S7).
The correlation coefficients are distributed aroundtwo distinct peaks at ~0.995 and ~0.96 (Fig. 2C).
The peaks with larger and smaller coefficients
correspond to the open (O) and closed (C) states,
respectively. On the other hand, the correlation
coefficient analysis for the nucleotide-free and
AMPPNP-bound a3b3 showed static conformations of b (figs. S8 and S9 and table S1). Figure
2D shows typical time courses of the two conformational states for individual b’s. In all images
observed in the presence of 2 to 4 mM ATP (n =8746 frames), three b’s at a given frame dominantly showed the CCO state (82% of total).
The COO state was also observed (14.5%). Other
states such as CCC (3%) and OOO (0.5%) wererare. We analyzed the rotary propagation of the O
and C states by counting the number of counterclockwise shifts of the CCO state [Fig. 2E andsupporting online material (SOM)]. The propa-

Cumulated number of CCO shifts

b’s retracted toward the center and simultaneously lowered their protrusions. Consequently,
the ring showed a single high protrusion. A simulated image of a3b3 with bound nucleotides wasconstructed using a structure in which g was removed from the crystal structure of F1 (Fig. 1E)(4). The simulated image (Fig. 1F) also showed
an asymmetric ring very similar to that of theobserved image. The excellent agreement indicates that only two b’s can assume the closed
conformation, even in saturating AMPPNP. Thisfeature is consistent with the observation that threeb’s do not assume the closed conformation simultaneously (22).
When ATP was added, b showed distinct
conformational dynamics; each b underwent a
conformational transition between the outwardly
extended high state (open) and the retracted low
state (closed) (Fig. 2A and table S1); the outwardly extended and retracted conformations
correlated well with the high- and low-protrusion
states, respectively (fig. S6). The most prominent
features are that only a single b assumes the openstate, as in the presence of AMPPNP, and thatwhen the open-to-closed transition occurs at oneb, the opposite closed-to-open transition occurs
simultaneously at its counterclockwise neighbor

757

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17. T. Ando et al., Proc. Natl. Acad. Sci. U.S.A. 98, 12468(2001).
18. T. Ando, T. Uchihashi, T. Fukuma, Prog. Surf. Sci. 83, 337(2008).
19. M. Shibata, H. Yamashita, T. Uchihashi, H. Kandori,
T. Ando, Nat. Nanotechnol. 5, 208 (2010).
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468, 72 (2010).
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22. R. I. Menz, J. E. Walker, A. G. Leslie, Cell 106, 331(2001).
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Structural Basis for Tail-AnchoredMembrane Protein Biogenesis by theGet3-Receptor Complex
Susanne Stefer,1* Simon Reitz,2* Fei Wang,3 Klemens Wild,2 Yin-Yuin Pang,2 Daniel Schwarz,4Jörg Bomke,4 Christopher Hein,1 Frank Löhr,1 Frank Bernhard,1 Vladimir Denic,3†Volker Dötsch,1† Irmgard Sinning2†
Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation,
and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered
to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interactingwith the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 incomplex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and
4.6 angstrom resolution. The structural data, together with biochemical experiments, show thatGet1 and Get2 use adjacent, partially overlapping binding sites and that both can bindsimultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes inGet3 that are required for TA protein insertion. These data suggest a molecular mechanism fornucleotide-regulated delivery of TA proteins.
ophisticated mechanisms have evolved totarget eukaryotic membrane proteins tothe correct membrane-surrounded organelle and to protect them from aggregation. Of
central importance are cytosolic factors that decode the targeting information of a signal sequenceby transient association and escort membraneproteins to their designated location (1, 2). Tailanchored membrane proteins (TA proteins) consist of a N-terminal soluble domain and a single

S

1
Institute for Biophysical Chemistry, Centre for Biomolecular
Magnetic Resonance, Goethe University, D-60325 Frankfurt
am Main, Germany. 2Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg,
Germany. 3Department of Molecular and Cellular Biology,
Harvard University, Northwest Laboratories, Cambridge, MA
02138, USA. 4Merck KGaA, Merck Serono, Molecular Interactions and Biophysics, D-64293 Darmstadt, Germany.

*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:irmi.sinning@bzh.uni-heidelberg.de (I.S.); vdoetsch@em.
uni-frankfurt.de (V.D.); vdenic@mcb.harvard.edu (V.D.)

758

C-terminal transmembrane domain (TMD) (3–5).
The C-terminal localization of their targeting signal requires release of the synthesized proteinfrom the ribosome before interaction with an insertion machinery (6). This precludes TA proteinsfrom cotranslational insertion mediated by thesignal recognition particle (SRP)–Sec61 translocon
system (7–9), which mainly targets membraneproteins with a N-terminal signal sequence. Although some studies suggest an unassisted insertion of TA proteins into the mitochondrial outermembrane (10, 11), most endoplasmic reticulum(ER)–destined TA proteins are thought to insert
by an energy-dependent process, which involves
several cytosolic factors (2, 12–17). In yeast, it
was shown that the adenosine triphosphatase(ATPase) Get3 is necessary for the biogenesis of
TA proteins (2, 18). Get3 is a dimeric protein witheach subunit comprising a nucleotide-binding
domain (NBD) and a methionine-rich a-helicaldomain that has been implicated in TA protein
binding (TA binding domain, TABD). Crystal

5 AUGUST 2011

VOL 333SCIENCE

Acknowledgments: We thank M. Tanigawara, H. Yamashita,
R. Hasegawa, and D. Okuno for technical help andmembers of the Noji and Ando laboratories for valuablecomments. This work was supported by Grants-in-aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology project ID nos.
18074005, 18201025, 21770168, 21681017, 2102301,
21107517, 22247025, and 20221006); the Knowledge
Cluster Initiative; and the Japan Science and Technology
Agency for Core Research for Evolutional Science andTechnology.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/333/6043/755/DC1
Materials and Methods
Figs. S1 to S12
Table S1
References (26–28)
Movies S1 to S8
11 March 2011; accepted 21 June 201110.1126/science.1205510

structures of Get3 have shown that the protein
switches between an open and closed state, depending on its nucleotide load. Whereas in the
apo and magnesium-free adenosine diphosphate (ADP) forms the open state is favored(19–23), ADP-Mg2+ and the nonhydrolyzable
ATP analog 5′-adenylyl-b,g-imidodiphosphate–
Mg2+ (AMPPNP-Mg2+) induce the closed state(19), which is further tightened up in the transition state of adenosine triphosphate (ATP) hydrolysis (20). The hydrophobic groove responsible
for TA binding seems fully assembled only in thetransition state (20). At the membrane, Get3 interacts with the two receptor proteins, Get1 and
Get2, which are essential for TA protein insertion(2). In order to understand the molecular mechanisms underlying Get3-dependent membrane
insertion of TA proteins, we determined crystal
structures of Get3 in complex with the soluble
domains of its membrane-bound Get1 and Get2
receptors and functionally characterized theinteraction in different nucleotide-bound states.
Get1 and Get2 are integral membrane proteins, and each comprises three TMDs (Fig. 1A).
In order to analyze the Get3-receptor interaction
in more detail, we performed flotation assaysof Get3 with microsomal membranes (RMs)(Fig. 1B) and insertion assays with the TA protein Sec22 [a soluble N-ethylmaleimide–sensitive
factor attachment protein receptor (SNARE)
protein] in the presence of different nucleotides(Fig. 1C) (2, 14, 15). Efficient TA protein insertion was observed in presence of ATP and ADP,
but not with adenosine 5′-O-(3-thiotriphosphate)(ATP-g-S)—in line with Get3 recruitment observed in the flotation assays. Get1-CD (residues
19 to 103) is the most conserved region of Get1
and predicted to adopt a coiled-coil structure(24) with two long helices (a1 and a2) extending the flanking transmembrane regions (Fig. 1A
and fig. S1A), similar to its human homolog,
the tryptophan-rich basic protein, WRB (25, 26).
Using a pull-down approach, we found that
detergent-solubilized Get1 efficiently binds toGet3 (fig. S2A). Additional truncation constructs

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