Báo cáo Y học: Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus pptx

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

domain
(subunits a
3
b
3
cde), with the catalytic sites for ATP
synthesis, and the membrane-embedded F
o
domain
(bacterial subunits ab
2
c
10–15
), with the sites for the
translocation of the ions. In ATP synthesis mode, the
F
o
motor converts the electrochemical ion gradient
into torque to force the F
1
motor to act as an ATP
generator, whereas, for ATP hydrolysis, F
1
converts
the chemical energy of ATP hydrolysis into torque
causing the F
o
motor to act as an ion pump (for
reviews, see [1–4]). Rotation of the asymmetric
Keywords

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

of the rotor. The data indicated very tight binding between the c
11
ring and
the ce complex, with an apparent K
d
value of approximately 7.4 nm. The
rotor assembly was primarily dependent on the interaction of the c ring
with the c subunit, and binding of the c ring to the free e subunit was not
observed. Mutagenesis of selected conserved amino acid residues of all
three rotor components (cR45, cQ46, cE204, cF203 and eH38) severely
affected rotor assembly. The interaction kinetics between the ce complex
and c
11
ring mutants suggested that the assembly of the c
11
ce complex was
governed by interactions of low and high affinity. Low-affinity binding was
observed between the polar loops of the c ring subunits and the bottom
part of the c subunit. High-affinity interactions, involving the two residues
cE204 and eH38, stabilized the holo-c
11
ce complex. NMR experiments
indicated the acquisition of conformational order in otherwise flexible
C- and N-terminal regions of the c subunit on rotor assembly. The results
of this study suggest that docking of the central stalk of the F
1
complex
to the rotor ring of F
o
to form tight, but reversible, contacts provides

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

[11], the c ring forms a tight rotor
complex with the F
1
subunits c and e, similar to the
tight binding between the stator components
(ab
2
a
3
b
3
d)ofF
1
and F
o
[11–13]. Although high-resolu-
tion structures exist for ce within the F
1
complex (e.g.
[14], ce complex [15], isolated e subunit [16] and the
c
11
ring from Ilyobacter tartaricus [10]), our knowledge
about the F
1
–F
o
rotor interaction is restricted to a
3.9 A
˚

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

o
, with
a K
d
value in the nanomolar range, and identify indi-
vidual contributions of important amino acid residues
in rotor complex stability. Further, we were able to
monitor the accretion of structural ordering within flex-
ible domains of the c subunit on rotor assembly.
Results
In vitro rotor assembly from the c
11
ring and
subunits c and e
The aim of this study was to obtain a better under-
standing of the binding processes of the rotor subunits
(c
11
, c and e) from I. tartaricus ATP synthase. For this
purpose, the membrane-embedded F
o
rotor part, the
c
11
ring, was used from wild-type I. tartaricus cells and
from I. tartaricus cells and from E. coli cells heterolo-
gously expressing the I. tartaricus c
11
ring ([25,26] and
Supporting information). The c and e subunits, form-

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

11
ring to the
c¢e complex studied by SPR
The kinetic characteristics of the interaction between
the c¢e complex (as a ligand) and the isolated c ring
(as an analyte) were studied in detail by SPR spectros-
copy with a Biacore instrument. A typical set of exp-
erimental kinetic traces recorded at different
concentrations of analyte is shown in Fig. 2A. At
higher c ring concentrations, a minor systematic devia-
tion was observed between the measured and fitted
curves, indicating a slow, probably nonspecific, binding
process (Fig. 2B). Association and dissociation rate
constants (k
on
and k
off
, respectively; see Eqns (1) and
(2) in Experimental procedures) were independent of
c ring concentration in the range 1–300 nm. These data
allowed us to calculate (K
d
= k
off
⁄ k
on
) a dissociation
equilibrium constant (or affinity constant) K
d
of about

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

2+
-nitrilotriacetic acid agarose with subsequent application of the c ring. The eluates were col-
lected and analysed by SDS-PAGE. The molecular masses and proteins used in this experiment are indicated on the left and right, respec-
tively. (A) 1, purified c¢e complex; 2, elution of the rotor in 1.5 m
M DHPC. (B) Reconstitution of the heterologously synthesized c rings with
the c¢e complex: 1, elution fraction with the wild-type c rings; flow through and elution fractions with the two c ring mutants cR45A (lanes 2
and 3, respectively) and cQ46E (lanes 4 and 5, respectively). (C) Purification of c¢F203A ⁄ e: 1, flow through; 2, wash; 3, elution. (D) Reconsti-
tution of the c rings with the c¢e complexes harbouring two point mutations: flow through and elution fractions of the experiments with the
mutants c¢E204A ⁄ e (lanes 1 and 2, respectively) and c¢eH38A (lanes 3 and 4, respectively). (E) Reconstitution of c rings with separate sub-
units c¢ and e: flow through and elution fractions of the experiments with the e-His (lanes 1 and 2, respectively) and His-c¢ (lanes 3 and 4,
respectively) subunits.
Time (s)
0 100 200 300
Response (RU)
0
500
1000
1500
2000
2500
3000
A
B
(1) 500 n
M
(2) 300 n
M
(3) 100 n
M
(4) 10 n

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

(5) 1 nM
Fig. 2. SPR binding and dissociation kinetics
of detergent (DHPC)-solubilized c ring to the
c¢e complex immobilized on an Ni
2+
-nitrilotri-
acetic acid surface. (A) Overlay plot showing
the concentration-dependent interaction
kinetics of the c ring at 500, 300, 100, 10
and 1 n
M, and the single exponential fitting
curves (bold) for association (black) and dis-
sociation (grey). (B) Representative residual
plot showing the deviation of the mathe-
matical fit relative to the data points.
Rotor interactions of the F-ATP synthase D. Pogoryelov et al.
4852 FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS
above 10 mm (Fig. 3A). A strong interaction was also
observed in the presence of Mg
2+
at concentrations
above 10 mm, and this c
11
–ce interaction could not be
distinguished from effects caused by other ions (K
+
,
Mg
2+
,Ca

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

off
), which was partially compensated for by a
fast association rate k
on
. At a higher pH, dissociation
stabilized at a slower rate, but, above pH 8.5, the
association rate was drastically reduced. Taken
together, the affinity constant remained relatively
unchanged over a wide range of pH, but decreased
significantly (higher K
d
) at values above pH 8.5. If a
high pH (9.5) was combined with a low salt condi-
tion, the association of the c ring with the c¢e com-
plex was impeded (not shown). This combination
essentially reproduced the well-documented F
1
–F
o
separation (or stripping) condition, which seems to be
caused by an impaired reassociation of F
1
with F
o
at
the rotor interface.
Mutations affecting the interaction of c
11
with c¢e
In order to assess the interaction of the c¢e complex

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

kinetics of c ring binding to the mutant c¢e complexes
(Fig. 6A). The rate constants k
on
and k
off
and the
Table 1. Summary of binding parameters. Binding parameters ⁄ constants for the interaction of c rings with immobilized wild-type (wt)
c¢e complex (A, B), c¢e complex with c¢ mutants (C), c¢e complex with e mutants (D) and individual c¢ subunits (E). Rate constants and K
d
were calculated as described in Experimental procedures. Data in A represent the mean ± standard deviation (SD) of 10 independent experi-
ments at five different ligand concentrations each; data in B–E represent the mean of two to three independent experiments at three differ-
ent concentrations each. Rate and affinity constants of the mutants are considered to be different from wt when differing by more than
1SD (wt).
Analyte Ligand k
off
· 10
)3
(s
)1
) k
on
· 10
4
(M
)1
Æs
)1
) K
d
(nM)

(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

significantly. The
k
on
values of both mutants decreased approximately
10-fold, and the k
off
values increased by two orders of
magnitude (Table 1C). Consequently, their dissociation
equilibrium constants (K
d
) were at least three orders of
magnitude higher than those obtained with the wild-
type c¢e complex. Therefore, the formation of rotor
complexes harbouring the c¢E204A mutant with a
reduced stability can be corroborated by the in vitro
reconstitution method (Fig. 1D, lanes 1 and 2). The
c¢E204K mutant showed only weak residual binding,
with k
on
and k
off
values at the detection limits, suggest-
ing that c¢E204 is the most critical of these acidic resi-
dues for the formation of a stable rotor complex. This
observation is corroborated by earlier work which
showed that replacement of the homologous residue in
E. coli (cE208) by K or C decreased the enzyme’s cou-
pling and proton pumping efficiency [22,28]. However,
the second site mutations in the hydrophilic loop of
the c subunits suppressed the uncoupling effect of

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

M,
pH 7). (B) pH dependence of the binding
association (k
on
) and dissociation (k
off
) rate
constants determined in 10 m
M BisTrisPro-
pane-HCl buffer (pH 5.5–9.5) in the pres-
ence of 300 m
M NaCl and 2 mM MgCl
2
.
Rotor interactions of the F-ATP synthase D. Pogoryelov et al.
4854 FEBS Journal 275 (2008) 4850–4862 ª 2008 The Authors Journal compilation ª 2008 FEBS
reconstitution experiments (Fig. 1E, lanes 3 and 4), as
well as SPR analyses (Fig. 4B), indicate that the c ring
binds to the separate c¢ subunit with an approximately
five-fold lower SPR response when compared with the
c¢e complex (Fig. 2A, Table 1E). This probably occurs
as a result of improper folding of the protein, in line
with the observed slight deviation of the binding and
dissociation kinetics. Binding of the c¢E204A mutant,
with or without complex formation, with the e subunit
to the c ring also shows a similar low range of K
d
(compare Fig. 4B, Table 1E with Fig. 6A, Table 1C).
The latter observation suggests that interaction studies
with the isolated c subunits may represent a feasible

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

d
: micromolar range; Table 1D). Moreover, the
A
B
Fig. 4. SPR binding and dissociation kinetics
of the c rings to the immobilized c¢e com-
plex and the separate subunits c and e. (A)
Heterologously expressed wild-type c ring
(broken line) and cR45 or cQ46 mutant (full
lines) at 100 n
M. (B) Kinetic traces of wild-
type c ring (500 n
M) to immobilized separate
subunits c (1–3) and e (4). The single expo-
nential fitting curves are depicted in bold for
association (black) and dissociation (grey)
phases. The c ring does not interact with
the c¢F203A mutant and the separate e sub-
unit. Binding of the c ring to the separate
c¢ unit seems to be more complex, involving
a more pronounced slow component.
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 4855
replacement of eH38 with K or D affected rotor
assembly so severely that the binding kinetics were
clearly too slow for quantitative analysis in our time
window.
NMR analysis of the interaction between the
c¢e complex and the c
11

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.

Figure 7 shows that 18 of the 28 cross-peaks assigned
to both N- and C-terminal loop regions of the c sub-
unit (residues 59–70 and 198–207, Fig. S1) were broad-
ened beyond detection when titrating the isolated
c¢e complex with the c oligomer. The maximum effect
of resonance broadening was observed at c
11
: c¢e
molar ratios of 1 : 1 and above, confirming a single
binding site between the interacting components, as
predicted by SPR analysis. The observed broadening
of the c¢ subunit resonances indicates that flexible
loops of the c¢ subunit become structured on binding,
reaching the spin relaxation rates of the entire com-
plex. Although the contribution of the c subunit could
be clearly shown by both techniques used in this work,
an involvement of the e subunit in rotor assembly
could only be pinpointed by SPR analysis (eE29,
eD31and eH38) because of the lack of signals in TRO-
SY.
Discussion
Two F
1
–F
o
binding affinities during rotor
assembly
We have shown that the interaction between the c ring
and the central stalk subunits c and e of the rotor of
I. tartaricus ATP synthase comprises high-affinity

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 c
11
ring and the c¢e complex can be distinguished
experimentally by a high-affinity interaction (nm) that
can be shifted to low-affinity binding (lm) by mutation
of the c¢E204 or eH38 residues to Ala. These residues
appear to be responsible for specific high-affinity con-
tacts with cR45 and cQ46; consequently, mutation of
c¢E204 or eH38 results in a fast dissociation of the
c ring from the c¢e complex. The isolated c¢ subunit
can achieve a high-affinity interaction with the c
11
ring
in the absence of the e subunit, although less robust
than with the c¢e complex. However, in the mutant
c¢E204A, only low-affinity binding is maintained, and
this is influenced by changes in the ionic strength and
pH. This is completely abolished by mutating selected
residues [cR45 (A,Q,Y,E), cQ46 (A,Y,E) and cF203A],
suggesting that the c subunit also contributes to the
establishment of the low-affinity contacts with the
c ring. Furthermore, our data suggest that the sepa-
rately synthesized e-His subunit does not interact with
the c ring by itself; only when complexed with the
c¢ subunit can the conserved eH38 establish a high-
affinity interaction. This is in agreement with data
from chloroplast and yeast mitochondrial ATP
synthase [33,34], where the rotor could be assembled
only from the c subunit and the c ring. However, in
contrast with E. coli and Bacillus PS3 ATP synthases,

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

binding was observed between the c ring
and c¢E204K (7) (A) or eH38K ⁄ D(7⁄ 8) (B).
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 4857
plasticity, between the F
1
and F
o
complexes, and the
switching between tight and weak affinity may play a
role in the coupling activity of the enzyme in some
ATP synthases. Presently, there is no experimental
evidence for this hypothesis, but it is well established
for chloroplast ATP synthases that the c subunit con-
tains a unique, 40-amino-acid regulatory domain at
the bottom of the c subunit (Fig. 5A), which is
involved in coupling of the enzyme via redox-thiol
modulation [33,39,40]. The approach of probing the
rotor’s interface, as established in this work, could
represent a feasible method to study further the cou-
pling and regulation between the F
1
and F
o
complexes
in these plant-type ATP synthases.
Structural considerations in the assembly and
interaction of rotor components
Figure S1 shows a model for the location of the resi-
dues involved in the high-affinity interaction (cE204

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-

tion requires further research.
In contrast with the c and e subunits, which have a
considerably high variation of amino acid residues in
the contact region with the c ring, multiple amino acid
sequence comparisons of c subunits from F-ATP syn-
thases show very high conservation of the loop amino
acids [R(K), Q, P, E(D)]. The surface structures of
these c ring loop regions, and their local charge distri-
bution [10] in particular, indicate that the contact sites
of all c rings comprise inner and outer rings with posi-
tive and negative charges, respectively (Fig. S1). This
A
B
Fig. 7. Solution NMR of the c¢e complex.
1
H,
15
N-TROSY-HSQC
spectra of the
2
H,
15
N-uniformly labelled c¢e complex in 3 mM
DHPC, 50 mM K
2
HPO
4
⁄ KH
2
PO

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

ATPase, but also to larger c rings from other species
(D. Pogoryelov and T. Meier, unpublished data; [46]).
This work demonstrates clearly that the c ring residues
cR45 and cQ46, which are part of the c ring loop
region, are both obligatory for high- and low-affinity
contacts with the c¢e complex, and that only c rings, but
not monomeric c subunits, can form a complex with c¢e.
The binding studies between the c¢e complexes and
the c
11
ring presented here represent an in vitro simula-
tion of complex assembly, and the limitations must be
emphasized. The conclusions drawn in this article are
based entirely on measurements with isolated subunits;
hence, in principle, they may not represent the situa-
tion during in vivo ATP synthase assembly. However,
our results are in accordance with the reported affinity
constants measured for the ATP synthase, contact
sites, cross-links and effects of critical point mutations
on the rotor assembly in the functional enzyme
([13,18–22,28–30,34,47–53] and references therein].
Hence, the rotor assembly observed in vitro in this
study could provide a glimpse into the in vivo forma-
tion of the native ATP synthase rotor and hence F
1
F
o
assembly. In our view, docking of the central stalk of
the F
1

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

and 2 mm MgCl
2
[buffer (1)]. The material on the column
was then equilibrated with buffer (1) containing one of the
selected detergents (1.5 mm DHPC, 0.02% DDM or 1%
OG), and 12 mL of the purified c ring sample [0.1 lm in
buffer (1) containing the same detergent] were applied.
Unbound c ring was removed by washing with three col-
umn volumes of buffer (1) containing the selected detergent,
and elution of the reconstituted rotor complex was per-
formed by the addition of two column volumes of elution
buffer containing 50 mm potassium phosphate (pH 7.0),
300 mm NaCl, 400 mm imidazole and 2 mm MgCl
2
and the
same detergent. The same procedure was used to check
complex formation of the c ring with the single isolated
subunits c¢ and e. All eluted rotor complexes were analysed
by SDS-PAGE.
SPR binding assays
Binding of the c ring to immobilized His-tagged c¢e com-
plexes, or to separate c¢ and e subunits, was studied quanti-
tatively using a BIACORE 2000 and nitrilotriacetic acid
sensor chip from Biacore AB (Uppsala, Sweden). The sur-
face was Ni
2+
coated with a 3 min injection of 1 mm
NiSO
4
at a flow rate of 10 lLÆmin

)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

traces, and was therefore selected for further studies.
The running buffer was 20 mm Tris ⁄ HCl pH 7.0,
300 mm NaCl, 50 lm Na
2
EDTA and 1.5 mm DHPC. This
composition was modified to account for specific experi-
mental needs, as otherwise specified (detergent, salt or pH).
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 4859
Binding experiments were performed at 20 °C at a constant
flow rate of 10 l LÆmin
)1
in the running buffer. The mea-
sured kinetics were not determined by diffusion limitations,
as higher flow rates (e.g. 30 lLÆmin
)1
) and lower analyte
immobilization levels had no obvious effect on the kinetics,
and the kinetic data could be fitted by single exponentials
(see below and Fig. 2). The injection of analyte (c
11
rings)
in running buffer was accomplished in 3 min (association
or contact phase), followed by another 3 min injection of
running buffer (dissociation phase). The nitrilotriacetic acid
chip surface was regenerated with a 3 min injection of strip
buffer (0.5% SDS, 50 mm Na
2
EDTA, pH 8.0) and rinsed
prior to injection of the next binding experiment. The

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

off
is the
dissociation rate constant (s
)1
), R
0
is the response at the
start of dissociation (RU), t
0
is the dissociation start time
(s) and R
offset
is the residual response at infinite time (RU).
The offset correction was used to correct the refractive
index change caused by rapid changes of unbound analyte
concentration at the end of the association phase. Dissocia-
tion phases were fitted in the time interval of 120 s after the
end of analyte (c ring) injection.
The association rates (k
on
) were determined from the
association kinetics in the time interval from 10 to 180 s fit-
ted to a model assuming a single phase ⁄ single binding site
interaction according to the equation:
y ¼ k
on
CR
max
=ðk
on

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

I
is the bulk refractive index effect (RU). A time interval
immediately following injection (10 s) was avoided because
of apparent mixing effects and large bulk refractive index
changes, as well as possible problems with mass transport
limitation at high concentrations of bound analyte [56]. The
dissociation constant K
d
was resolved by the equation
K
d
= k
off
⁄ k
on
. For the wild-type interaction of the c ring
with the c¢e complex, this was independently cross-checked
by analysis of equilibrium data (Scatchard plot), yielding a
K
d
value of the same order of magnitude.
Acknowledgements
We thank Gregory Cook for reading the manuscript.
This work was supported by the Cluster of Excellence
‘Macromolecular Complexes’ at the Goethe University
Frankfurt (DFG Project EXC 115).
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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