Probing the rotor subunit interface of the ATP synthase
from Ilyobacter tartaricus
Denys Pogoryelov
1,2
, Yaroslav Nikolaev
3,
*, Uwe Schlattner
4,5
, Konstantin Pervushin
3,
,
Peter Dimroth
1
and Thomas Meier
1,2
1 Institute of Microbiology, Eidgeno
¨
ssische Technische Hochschule, Zurich, Switzerland
2 Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany
3 Laboratory of Physical Chemistry, Eidgeno
¨
ssische Technische Hochschule, Zurich, Switzerland
4 Institute of Cell Biology, Eidgeno
¨
ssische Technische Hochschule, Zurich, Switzerland
5 Laboratory for Fundamental and Applied Bioenergetics, Inserm E0221, University Joseph Fourier, Grenoble, France
F-ATP synthases convert the energy of an electro-
chemical proton or sodium ion gradient into ATP, the
universal chemical energy source of living cells. These
enzymes are composed of a water-soluble F
1

c ring; F
1
F
o
ATP synthase;
Ilyobacter tartaricus; rotor subunit
interaction; surface plasmon resonance
Correspondence
T. Meier, Max-Planck Institute of
Biophysics, Max-von-Laue Str. 3, 60438
Frankfurt am Main, Germany
Fax: +49 69 63033002
Tel: +49 69 63033038
E-mail: thomas.meier@mpibp-frankfurt.
mpg.de
Present addresses
*Biozentrum, University of Basel,
Switzerland
School of Biological Sciences, Nanyang
Technological University, Singapore; Biozen-
trum, University of Basel, Switzerland
(Received 8 February 2008, revised 29 July
2008, accepted 1 August 2008)
doi:10.1111/j.1742-4658.2008.06623.x
The interaction between the c
11
ring and the ce complex, forming the rotor
of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon
resonance spectroscopy and in vitro reconstitution analysis. The results pro-
vide, for the first time, a direct and quantitative assessment of the stability

an explanation for the relative ease of dissociation and reconstitution
of F
1
F
o
complexes.
Abbreviations
DDM, n-dodecyl b-
D-maltoside; DHPC, dihexanoylphosphatidylcholine; HSQC, heteronuclear single quantum correlation; OG, octyl
b-
D-glucoside; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; RU, response unit; SPR, surface plasmon resonance; TROSY, transverse
relaxation-optimized NMR spectroscopy.
4850 FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS
c subunit within the hexameric assembly of alternating
a and b subunits elicits conformational changes in the
catalytic b subunit sites, resulting in ATP synthesis,
consistent with the ‘binding change model’ [5], the
crystal structure of F
1
[6] and single-molecule video
microscopy [7].
In an ATP synthase at work, drag is imposed by the
F
1
motor components; this has been proposed to cause
elastic energy storage within the a-helical domain of
the c subunit [8], the peripheral stalk [9] and the rotat-
ing c ring [10]. To withstand the resulting strain of up
to – 55 kJÆmol
)1

resolution F
1
c
10
structure from yeast ATP syn-
thase [17]. On the basis of these structures, the lower
part of the F
1
complex can be derived at a resolution
suitable for the identification of possible amino acid
residue candidates forming the interface between ce
and the c ring, and these residues have been corrobo-
rated by cross-linking experiments and EPR spectro-
scopy of site-directed spin labels. Using these
approaches, the e subunit residues 26–33 and 38 (Esc-
herichia coli numbering) [18–20] and the c subunit resi-
dues 200–210 [21,22] are localized in the direct vicinity
of the hydrophilic loop units of the c ring.
In this article, we have used surface plasmon reso-
nance (SPR) [23] and NMR spectroscopy [transverse
relaxation-optimized NMR spectroscopy (TROSY) and
NOE-TROSY [24]] to obtain a greater understanding
of the interaction sites and affinities between the
ce complex and the c
11
ring during the assembly of the
I. tartaricus ATP synthase. We report tight, but revers-
ible, binding between the rotor parts of F
1
and F

ing the water-soluble F
1
rotor complex of the I. tar-
taricus ATP synthase, were heterologously expressed in
E. coli cells: we constructed appropriate expression
vectors for the synthesis of His-tagged c and e sub-
units, and purified individual c¢ (residues 12–253 [15])
and e subunits, and the c¢e pair, by Ni
2+
-nitrilotriace-
tic acid affinity chromatography (Fig. 1A, lane 1). To
assess rotor assembly, the c
11
ring was applied to the
c¢e complex on the surface of the Ni
2+
-nitrilotriacetic
acid resin of the column, and the c
11
c¢e complex
(rotor) was eluted by increasing the imidazole concen-
tration (Fig. 1A, lane 2). This method yielded stable
rotor complexes in the presence of several non-ionic
detergents, e.g. dihexanoylphosphatidylcholine (DHPC),
octyl b-d-glucoside (OG) and n-dodecyl b-d-maltoside
(DDM) (shown for DHPC in Fig. 1A, lane 2). The
in vitro formation of these rotor assemblies was further
corroborated by native gel electrophoresis and gel
filtration experiments (data not shown).
Binding characteristics of the c

7nm based on 50 independent binding experiments
under standard conditions, with individual experimen-
tal values scattering in the range 4.1–10.7 nm
(Table 1A). Thus, the high-affinity interaction between
the c ring and the c¢e complex is characterized by a
very slow dissociation. Such an affinity is comparable
with that of a typical antigen–antibody complex [27],
and consistent with that published for the E. coli
F
1
F
o
complex [13]. The parameters of all the interac-
tions that could be quantified are summarized in
D. Pogoryelov et al. Rotor interactions of the F-ATP synthase
FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS 4851
Table 1. In binding experiments using monomeric
c subunits, we could not detect any interaction with
the c¢e complex (data not shown).
With respect to salt, binding of the c ring to an
immobilized c¢e complex was weak at NaCl concentra-
tions below 500 lm and strong at NaCl concentrations
AB C D E
Fig. 1. SDS-PAGE showing the purification and reconstitution experiments of rotor subunits (c
11
c¢e) from I. tartaricus ATP synthase. The
rotor subunits c¢, e and the c
11
ring were purified as described in the Supporting information. Reconstitution was performed by binding the
His-tagged subunits (either His-c¢ or e-His) to Ni

M
(5) 1 n
M
Fit k
on
Fit k
off
(1)
(2)
(3)
(4)
(5)
Time (s)
020 6040 80 100 120 140 160
Response (RU)
–100
–50
0
50
100
(1)
(2)
(3)
(4)
(5)
(1) 500 n
M
(2) 300 nM
(3) 100 nM
(4) 10 nM

2+
,Cl
)
and SO
4
2)
) at concentrations above
10 mm, indicating that the binding strength was depen-
dent on the ionic strength of the buffer and not on a
specific ion (e.g. Mg
2+
). Therefore, the specific require-
ment of Mg
2+
for the assembly of F
1
and F
o
into a
functional entity could not be attributed to these con-
tact sites at the rotor interface.
The pH of the solution, however, had a significant
impact on the rate constants k
on
and k
off
of the inter-
acting partners (Fig. 3B). A low pH (5.5) favoured
fast dissociation of the c ring from the c¢e complex
(high k

with selected amino acids in the loop region of the iso-
lated c ring [amino acids RQPE(D)], we introduced
point mutations at position cR45 or cQ46 and isolated
the corresponding c rings (Fig. 1B, lanes 2 and 4). The
interactions of the stable c rings with the c¢e complex
are shown in Fig. 4A. Strong binding was observed for
the heterologously synthesized wild-type c rings, with
rate constants (k
on
and k
off
) and derived dissociation
equilibrium constants (K
d
) almost identical to those
obtained with the c ring isolated from I. tartaricus cells
(Table 1B). Mutant c rings (R45A, Q, Y and E; Q46A,
Y and E) did not bind to the c¢e complex, as revealed
by SDS-PAGE (Fig. 1B) and SPR kinetic analysis
(Fig. 4A). The mutants cP47A and cE48A did not
form c ring complexes sufficiently stable for isolation
(not shown).
The contact region of the c¢e complex to the polar
loop of the c subunit can be allocated to the E. coli
c subunit residues 200–210 [21,22]. An amino acid
sequence alignment of this c subunit region (Fig. 5A)
shows low sequence conservation, but some acidic resi-
dues are abundant. We replaced each of these residues
(c¢E197, c¢E204, c¢E208 and c¢D209, I. tartaricus num-
bering) individually by Ala and determined the SPR

(A) wt c ring wt c¢e 1.1 ± 0.1 14.9 ± 3.2 7.4 ± 3.3
(B) wt c ring, recombinant wt c¢e 1.1 9.9 11.1
(C) wt c ring c¢D209A ⁄ e 0.8 8.6 10.1
c¢E208A ⁄ e 0.9 9.7 10.5
c¢E197A ⁄ e 1.1 8.6 14.3
c¢Y201A ⁄ e 2.0 8.6 21.9
c¢E204A ⁄ e 76.7 0.5 16300
c¢E204Q ⁄ e 78.5 0.6 12800
(D) wt c ring c¢eD31A 2.0 7.2 27.8
c¢eD31K 5.5 7.5 66.4
c¢eE29K 5.5 5.8 94.1
c¢eE29A 6.5 8.1 80.3
c¢eH38A 69.8 1.1 6600
(E) wt c ring c¢WT 1.5 7.5 19.7
a
c¢E204A 59.9 0.3 20800
a
This interaction is not entirely well described by a single exponential fit.
D. Pogoryelov et al. Rotor interactions of the F-ATP synthase
FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS 4853
derived dissociation equilibrium constants (K
d
) of the
c¢E197A, c¢E208A and c¢D209A mutants were in the
range of those determined for the wild-type c¢e com-
plex (Table 1C). In contrast, the two c¢E204 (A or Q)
mutations affected both k
on
and k
off

cE208K [28].
In addition to the negatively charged residues, the
flexible loop at the bottom of the c subunit also con-
tains two aromatic residues (cY201 and cF203), which
are conserved in bacterial ATP synthases (Fig. 5A).
The results of SPR analysis of the complex formation
for the c¢Y201A mutant (Fig. 6A, Table 1C) showed
only minor changes in the affinity, but the c¢F203A
mutant prevented the formation of a stable c¢e com-
plex (Fig. 1C) and only weak binding between the
c ring and c¢F203A was detected (Fig. 4B), in agree-
ment with functional studies made with the homolo-
gous amino acid residue Y205 in the c subunit of
E. coli [29,30]. It may be noteworthy that in vitro
A
B
Fig. 3. Effect of salt and pH on the binding
of the c ring (100 n
M) to immobilized
c¢e complex. (A) Dependence of the equilib-
rium response (R
eq
) on the salt (NaCl) con-
centration in the binding buffer. The values
for R
eq
were derived from the contact phase
fit of the corresponding experimental kinetic
traces. The binding experiments were per-
formed in BisTrisPropane-HCl buffer (2 m

approach for selected cases.
Influence of the e subunit on the stability of the
rotor
In contrast with the separate c subunit, a specific inter-
action of the c ring with a separate e subunit could
not be observed by SPR analysis (Fig. 4B) or in vitro
reconstitution (Fig. 1E, lanes 1 and 2). To investigate
whether the e subunit has an auxiliary role in rotor
assembly, interaction kinetics with the e subunit
mutants were recorded. The results in Fig. 6B and
Table 1D show that the replacement of eE29 or eD31
with A or K (numbering is equivalent in E. coli and
I. tartaricus) increased the dissociation rate of the
c ring from the c¢e complex by about two- to six-fold,
but the association rates remained largely unchanged.
The resulting increased K
d
value (i.e. lower affinity)
indicates a contribution of residues eE29 and eD31 to
rotor stability, and is in good agreement with previous
work, which showed partial uncoupling of the E. coli
ATP synthase by the mutations eE29, eD31 and eH38
[18–20,31]. A substantial alteration in the assembly of
the rotor was observed in the mutant eH38A (Fig. 6B),
resulting in an approximately 10-fold decrease in k
on
and increase in k
off
by almost two orders of magnitude
(K

ring
To investigate the interaction between the isolated
c¢e complex and the detergent-solubilized c
11
ring by
NMR spectroscopy, we employed conventional stable
isotope (
2
H ⁄
15
N ⁄
13
C) labelling techniques, as well as
TROSY-heteronuclear single quantum correlation
(HSQC) and three-dimensional (3D)-TROSY-
HNCA ⁄ HNCACB pulse schemes [32] (for assignment
and data interpretation, see Supporting information).
Titration experiments were performed using the
2
H,
15
N-labelled c¢e complex with an unlabelled c ring
solubilized in DHPC micelles. All changes in the
1
H,
15
N-TROSY-HSQC spectra were attributed solely
to the interaction of the c¢e complex with the c oligo-
mer, as no changes in the c¢e spectra were observed
when adding detergent micelles without protein.

A
B
Fig. 5. Protein sequence alignments of amino acid stretches structurally located at the F
1
–F
o
interface of the central stalk domain of F-ATP
synthases. The sequence alignments of subunit c (A) and subunit e (bacteria) ⁄ d (eukaryotes) (B) include species for which high-resolution
structures are available (comprising the amino acid stretches of interest). Secondary structures are shown on top of the alignments (bacteria,
full line; eukaryotes, broken line). The numbering is according to the sequence of I. tartaricus. Conserved amino acids [57] are in bold. Resi-
dues which have been characterized by F
1
–F
o
cross-links (for references, see Introduction) are underlined. The conserved charged and aro-
matic amino acid residues attributed to the rotor interface are highlighted (in black or grey, respectively). The critical residues for the
interaction of the c¢e complex with the c ring are marked by an asterisk.
Rotor interactions of the F-ATP synthase D. Pogoryelov et al.
4856 FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS
binding (K
d
% 7.4 nm). This value is similar to the
binding affinity determined in the stator complex
(ab
2
F
1
) of the E. coli ATP synthase [13]. Hence, rotor
and stator appear to contribute equally to the intrinsic
binding energy of complex assembly. The assembly of

the e subunit is essential for functional reconstitution
of F
1
with F
o
[20,35–38], but the partial contribution
of the e subunit to the stability of the rotor in these
cases is not yet clear.
Does the interaction of the c ring with the c and
e subunits have anything to do with the regulation of
enzyme activity? Potentially, this may be so. The low
and high affinities within the c
11
ring and ce complex
demonstrate not only a high stability, but also a high
A
B
Fig. 6. SPR kinetic traces of the interaction
between the wild-type c ring and c¢e com-
plexes carrying mutations in the c subunit
(A) and e subunit (B). Overlay plot showing
the SPR kinetics together with the single
exponential fitting curves (bold) for associa-
tion (black) and dissociation (grey). The
c ring concentration was varied from 10 to
500 n
M; only the SPR kinetics recorded at
300 n
M of the c ring are shown. Mutations
mainly affect the dissociation kinetics. No

and eH38), which is based on the structure of the cor-
responding complex from E. coli [15]. Both residues
are at the bottom of the ce complex and in close prox-
imity to each other. In the available structures of
c subunits from different organisms [14,15,38,41–44],
the amino acid stretch (residues 198–207, I. tartaricus
numbering) of the putative F
1
–F
o
interface falls into
the flexible region of the c subunit loop including resi-
dues cE(D)204 and cF(Y)203. These are the only con-
served residues in this stretch (Fig. 5A), and are
critical for the rotor stability as shown in this work.
According to our NMR spectroscopy data (Fig. 7),
this flexible region of the c subunit undergoes struc-
tural rearrangements in concert with the stretch of
residues 59–70, and they both become stabilized on
high-affinity interaction with the DHPC-solubilized
c
11
ring. The involvement of residues 59–70 from the
c subunit for complex formation with the c
11
ring has
not been detected previously [21] and, according to the
available structures of the c¢e complex, this loop is not
located at the predicted interface region. Therefore, a
possible involvement of this region in complex forma-

4
pH 7.0, 300 mM NaCl, 2 mM
MgCl
2
and 10% D
2
O, recorded at 5 °C and 600 MHz for 12 h. (A)
HSQC spectra of the c¢e complex (30 l
M). (B) HSQC spectra of c¢e
on addition of equimolar amounts of unlabelled c
11
ring. Numbering
corresponds to the resonances attributed to the individual amino
acid residues stemming from the c¢ subunit. Assignment (according
to the numbering of the I. tartaricus c subunit): 1, cG59; 2, cG70;
8, cE191; 9, cI190; 17, cE204; 21, cR192; 28, cV193. Inset in (A)
indicates the changes in the HSQC spectrum of the c¢e complex by
mutating the cE204 residue to Gln. Inset in (B) indicates the
changes in the selected areas of the HSQC spectrum of the
c¢e complex imposed by the addition of unlabelled c
11
ring at differ-
ent molar ratios.
Rotor interactions of the F-ATP synthase D. Pogoryelov et al.
4858 FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS
appears to be a common feature in all F-ATP synthas-
es, and this arrangement seems to be mandatory for
the formation of stable hairpin folding of the two heli-
ces of the c subunit [45]. Moreover, the c¢e complex is
able to bind not only to c rings from its native

complex to the rotor ring of F
o
to form tight,
but reversible, contacts must be one of the last steps in
the assembly of the ATP synthase complex, and can
explain the relative ease of dissociation and reconstitu-
tion of F
1
F
o
complexes observed more than four dec-
ades ago [54], and well documented ever since.
Experimental procedures
The construction of the plasmids, the synthesis and purifi-
cation of the subunits (c¢, e and c rings) and NMR meth-
ods are described in Supporting information.
In vitro reconstitution of the rotor complex
The whole reconstitution procedure was performed at
20 °C. The imidazole concentration of the c¢e sample was
first decreased to 40 mm by diluting the purified protein
(see above) 10 times with buffer containing 50 mm potas-
sium phosphate (pH 7.0), 300 mm NaCl and 2 mm MgCl
2
.
Then, 1 nmol of the material was immobilized on a 1 mL
Ni
2+
-nitrilotriacetic acid agarose column and washed with
three column volumes of 50 mm potassium phosphate buf-
fer (pH 7.0) containing 300 mm NaCl, 50 mm imidazole

)1
. About 1000 response
units (RUs) of ligand (purified His-tagged proteins diluted
in running buffer to 200 nm) were immobilized on the
nitrilotriacetic acid chip. This binding capacity gave an
optimal ratio between the specific signal (protein binding to
loaded chip) and nonspecific binding signal (protein and
detergent binding to empty chip), allowing the elimination
of the latter by baseline correction (see below). As a result
of the location of the His tag on the very top of the c¢ sub-
unit, the immobilized c¢e complexes were oriented upside-
down on the nitrilotriacetic acid surface of the chip, with
the bottom part of the c¢e complex exposed to the bulk.
Contaminating metal ions in the running buffer and
ligand buffer can influence the binding of the ligand to the
Ni
2+
-nitrilotriacetic acid surface. To increase the assay sta-
bility without influencing the dissociation rate of the ligand
from the surface, 50 lm of EDTA was added to all buffers
[55].
Association kinetic traces were recorded when c rings in
detergent containing buffer or reconstituted into 1-palmi-
toyl-2-oleoylphosphatidylcholine (POPC) liposomes were
passed over the loaded chip surface. In pilot SPR binding
studies, c rings reconstituted into POPC liposomes and
c rings solubilized in several detergents suitable for in vitro
reconstitution experiments were tested. DHPC was found
to cause negligible nonspecific binding to the immobilized
c¢e complex and good reproducibility of the SPR binding

background dissociation of immobilized ligand from the
Ni
2+
-nitrilotriacetic acid surface and nonspecific detergent
binding were monitored as changes in the signal (RU) dur-
ing blank runs (no analyte added to binding buffer) prior
to each binding experiment. The blank run traces were later
used for the baseline correction of the kinetic traces. Bind-
ing experiments were performed at several different concen-
trations of analyte (500, 300, 100, 10 and 1 nm) to calculate
reliable rate and affinity constants. The binding experiments
at each concentration of analyte were performed at least in
triplicate.
Kinetic analysis
On and off kinetics were analysed with biaevaluation 4.1
software. The dissociation rates (k
off
) were determined from
the dissociation kinetics of the sensograms fitted to (using)
the single-phase dissociation equation:
y ¼ R
0
exp½Àk
off
ðt À t
0
Þ þ R
offset
ð1Þ
where y is the response (RU), t is the time (s), k

C þ k
off
Þ
Âf1 À exp½Àðk
on
C þ k
off
Þðt À t
0
Þg þ R
I
ð2Þ
where k
on
is the association rate constant (m
)1
Æs
)1
), R
max
is
the maximum analyte binding capacity (RU), C is the
molar analyte concentration (m), t
0
is the injection start
time (s), k
off
is the dissociation rate constant (s
)1
) and R

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Supporting information
The following supplementary material is available:
Fig. S1. Modelling the F
1
–F
o
rotor interaction site of
the I. tartaricus F-ATP synthase.
Doc. S1. Supplementary results.
Doc. S2. Supplementary experimental procedures.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supplementary
material supplied by the authors. Any queries (other
than missing material) should be directed to the
corresponding author for the article.
Rotor interactions of the F-ATP synthase D. Pogoryelov et al.
4862 FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS