Structure and Flexibility of the C-Ring in the
Electromotor of Rotary FoF1-ATPase of Pea Chloroplasts
Shai Saroussi1., Maya Schushan1., Nir Ben-Tal1, Wolfgang Junge2, Nathan Nelson1*
1 Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel, 2 Division of Biophysics, University of
Osnabrück, Osnabrück, Germany
Abstract
A ring of 8–15 identical c-subunits is essential for ion-translocation by the rotary electromotor of the ubiquitous FOF1ATPase. Here we present the crystal structure at 3.4Å resolution of the c-ring from chloroplasts of a higher plant (Pisum
sativum), determined using a native preparation. The crystal structure was found to resemble that of an (ancestral)
cyanobacterium. Using elastic network modeling to investigate the ring’s eigen-modes, we found five dominant modes of
motion that fell into three classes. They revealed the following deformations of the ring: (I) ellipsoidal, (II) opposite twisting
of the luminal circular surface of the ring against the stromal surface, and (III) kinking of the hairpin-shaped monomers in
the middle, resulting in bending/stretching of the ring. Extension of the elastic network analysis to rings of different cnsymmetry revealed the same classes of dominant modes as in P. sativum (c14). We suggest the following functional roles for
these classes: The first and third classes of modes affect the interaction of the c-ring with its counterparts in FO, namely
subunits a and bb’. These modes are likely to be involved in ion-translocation and torque generation. The second class of
deformation, along with deformations of subunits c and e might serve to elastically buffer the torque transmission between
FO and F1.
Citation: Saroussi S, Schushan M, Ben-Tal N, Junge W, Nelson N (2012) Structure and Flexibility of the C-Ring in the Electromotor of Rotary FoF1-ATPase of Pea
Chloroplasts. PLoS ONE 7(9): e43045. doi:10.1371/journal.pone.0043045
Editor: Rajagopal Subramanyam, University of Hyderabad, India
Received June 8, 2012; Accepted July 16, 2012; Published September 25, 2012
Copyright: ß 2012 Saroussi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Israel Science Foundation Grant 204-10 (ISF) and by the United States–Israel Agricultural Research and Development
Fund (BARD) Research grant No. IS-4229-09. S.S. was supported by the Eshkol Foundation, the Israeli Ministry of Science. M.S. was supported by the Edmond J.
Safra Bioinformatics program at Tel-Aviv University. N.B.-T. acknowledges the support of grant 1331/11 from the Israel Science Foundation. Eshkol Foundation, the
Israeli Ministry of Science URL: http://www.most.gov.il/nr/exeres/8F309A05-C3FC-498D-AF16-DB2456D8AC4A.htm, Edmond J. Safra Bioinformatics program URL:
http://safrabio.cs.tau.ac.il/, Israel Science Foundation grant 1331/11 URL: http://www.isf.org.il/english/default.asp. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: nelson@post.tau.ac.il
. These authors contributed equally to this work.
comprising two b-type subunits; the dimer connects Fo with F1 (in
chloroplasts this dimer is a bb’ heterodimer). Despite being bound
to abb’, the c-ring is able to rotate relative to these subunits.
Subunit a hosts an essential arginine residue, which faces the
essential glutamic acid residue situated at the center of a proximate
hairpin-shaped monomer of c-subunit. Subunit a also contains two
proton half-channels connecting the acidic residue on the csubunit with the lumen and the stroma phases. Brownian
fluctuations, together with the ion-driven rotation of the c-ring
relative to the two half-channels on subunit a, are responsible for
generating the torque associated with rotary proton translocation
(see [5,13,14]). The stepped rotary motion of the c-ring relative to
the stator has recently been resolved experimentally for the
Escherichia coli enzyme [15,16]. The F1 portion of the ATP synthase
is characterized by a 3-fold stepping rotation; the mismatch
between this rotation and the 8-to-15-fold stepping of the Foportion of the ATP synthase is buffered by an elastic power
transmission between these motors. For the E. coli enzyme, the
stiffness of the stator [17] and a great elastic compliance of the
rotor have been determined (see [18,19,20,21], reviewed in [5]).
These functional features of the FoF1-ATPase are probably shared
by the V-ATPase, which is characterized by similar structures
[22].
Introduction
ATP (adenosine tri-phosphate), the general energy currency of
the cell, is supplied mainly by FOF1-ATPase (ATP synthase). This
enzyme is composed of two rotary machines, FO and F1, which are
coupled by a central rotor and a peripheral stator. FO translocates
ions (mostly protons) and generates torque at the expense of the
ion-motive force, and F1 synthesizes ATP at the expense of the
mechanical torque provided by FO [1,2,3,4,5]. The construction
principles of this enzyme have been conserved across a range of
species and structures spanning eubacteria, mitochondria and the
chloroplasts of higher organisms. A homo-oligomeric ring of the
hairpin-shaped subunit c is included in the Fo domain. The
number of copies of c subunits in this ring varies across organisms
and ranges from 8 (bovine mitochondria [6]) to 15 (cyanobacteria
[7]). In other words, the ion-to-ATP machines of different
organisms operate with different sized ‘gears’. Researchers agree,
however, that in a given organism the number of copies of subunit
c is constant, independent of the metabolic state [8,9]. The FoF1ATPase in chloroplasts, CFoCF1 [1,10], is the subject of this work.
The c-ring of the enzyme’s CFo portion consists of 14 copies of
subunit c [11,12], and is embedded in the membrane. The ring is
attached to a complex consisting of subunit a and a dimer
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C-Ring Structure and Flexibility
(DDM, Glycon, Inc.). The preparation was further purified by 2
steps of differential precipitation using PEG 2K as a precipitate
using 7% and 9%, respectively. The pellet was dissolved with
20 mM Tricine-Tris (pH 7.4), 0.125 mM dithiothreitol (DTT),
and 0.05% DDM, was applied to a 10–40% sucrose gradient
containing the same buffer, and was centrifuged using the SW-40
rotor (Beckman Inc.) at 37,000 rpm for 16 h. Fractions containing
CFoCF1 were pooled together and loaded onto an ion exchange
column (DEAE-cellulose, DE52, Whatman, Inc., 1.5618 cm) preequilibrated with 20 mM Tricine-Tris (pH 7.4), 0.125 mM DTT
and 0.05% DDM. The column was washed with 40 ml of the
same buffer, and fractions containing CFO dominated with
subunit-c were eluted by 50–250 mM NaCl linear gradient in
20 mM Tricine-Tris (pH 7.4), 0.125 mM DTT and 0.05% DDM.
These fractions were then pooled together and concentrated to 3–
4 mg/ml by 2 steps of differential precipitation using PEG 6K as
precipitant. The pellet was suspended in 5 mM Tris pH 8.0 and
0.05% Fos-choline 12.
The previously determined crystal structures of subunit c have
provided important insight into the role of the c-ring in the proton
translocating mechanism. In a given organism, the c-ring,
composed of 8–15 monomers as noted above, encompasses an
ion-binding site approximately in the ring’s middle plane
[6,12,23,24,25,26,27]. The functional acidic residue (mostly
glutamate, in some organisms aspartate) is situated at the
outward-facing helix of the hairpin. It is stabilized by hydrogen
bonds with neighboring residues. The dynamics of the ion-binding
site have been simulated by molecular dynamics (MD), revealing
structural transitions at the nanosecond time scale upon glutamate
deprotonation/protonation [28,29]. However, MD simulations
have been restricted to a narrow time window of 10–100 ns and
have not revealed large-scale motion in micro- to milliseconds as
relevant in the present case: When FO is decoupled from the F1portion, its unitary conductance is approximately 10 fS (for
chloroplasts see [30], for Rhodobacter capsulatus [31], and for E. coli
[32]), which implies about 1,000 rounds per second of the c-ring
(at 200 mV driving force).
Simple techniques for coarse-grained simulations of protein
dynamics, e.g., elastic network analysis, take just the Ca-atom of
each residue into account, and the solvent is usually neglected.
Such an approach does not require extensive computational power
and may, in principle, cope with micro- to millisecond events [33].
This restricted approach does not account for the over-damped
character of protein dynamics. More advanced techniques have
included solvent–protein interactions, damping and thermal
properties (for an application to adenylate kinase see [34]);
however, simulations are still carried out on a relative and not on a
real time scale.
Herein we applied normal mode analysis (without damping) to
the c-ring of Fo. The purpose was to identify the basic deformation
modes of the c-ring, and to investigate whether and how the
dominant elastic modes might contribute to the function of the
ring in this rotary ion-translocator. The method relies on the
position of the Ca-atoms embedded in an elastic network (in vacuo)
with a single force constant between neighboring nodes. Orthogonal eigen-modes of motion are computed [33]. For various
proteins, including membrane channels and transporters, the
slowest (i.e., global or cooperative) modes of motion have been
shown to represent functionally significant movements [33,35].
The isotropic Gaussian Network Model (GNM) has been used to
assess residue fluctuations and inter-residue dynamical correlations
[36,37], and the Anisotropic Network Model (ANM) has been
used to identify motion directionality in three dimensions (3D)
[38].
We isolated the c-ring of the FOF1-ATPase of the green pea
(Pisum sativum) from a native preparation, crystallized it, and
determined its homo-tetradecameric structure at a resolution of
3.4Å. The elastic eigen-modes of the ring structure were
determined by GNM and ANM, and the same analysis was
applied to c-rings from other FoF1-ATPases containing 8 to 15
copies of subunit c, respectively, and to a ring of a V-ATPase. We
discuss the roles of the dominant eigen-modes in rotary proton
translocation, in the stepped torque generation, and in the elastic
buffering of the stepped rotation for smooth torque transmission
into F1.
Crystallization and structure determination
Subunit-c crystals grew in a crystallization buffer containing
100 mM Na-acetate (pH 4.5), 50 mM MgCl2, 50 mM NaCl,
10 mM yttrium chloride, and 14–24% PEG 550 monomethyl
ether (MME), reaching their maximal size after 5–7 days. Yttrium
was added using a commercial additive kit (Hampton Research) as
it improved the shape of the crystals. After the crystals were
equilibrated and then incubated for 48 hours at 20uC, the quality
of the crystals and the subsequent diffraction pattern were
improved by adding to the reservoir 40% PEG 550 MME. The
crystals were then flash frozen in liquid nitrogen. Data were
collected in ID23-1, ESRF, Grenoble and processed by XDS [40].
Due to anisotropic diffraction, ellipsoid truncation of the data was
performed using the UCLA MBI Diffraction Anisotropy Server
[41]. Resolution limits were 3.7, 3.6 and 3.4Å along a*, b* and c*
axes, respectively. Initial phases were determined by maximumlikelihood molecular replacement as implemented in Phaser
[42,43], using a backbone model derived from the crystal structure
of the S. platensis c-ring (PDB ID 2wie [24]) as a search model.
Iterative refinement (PHENIX [44]) and manual building (Coot
[45]) using translation libration screw-motion (TLS) and noncrystallographic symmetry constraints using all fourteen monomers composing the ring produced a model at 3.4Å resolution with
R-work/free of 29% and 32%, respectively, with no outliers in the
Ramachandran plot. The structure was deposited in the Protein
Data Bank under the PDB ID 3V3C.
Sequence, evolutionary conservation and pKa
calculations
Using the sequence of subunit-c from the green pea, we initiated
a BLAST [46] search against the UniRef90 database [47],
collecting 351 sequences with Evalue ,0.0001. The full sequences
were then aligned using MAFFT [48] and input into the ConSurf
webserver (http://consurf.tau.ac.il, [49]) to compute conservation
scores. The conservation profiles were then mapped onto the cring structure.
The sequences of the c-rings with available structures were
aligned using the 3D-Coffee software package, and the alignment
process was further guided by the structural data [50]. Slight
manual adjustments were performed in order to achieve better
compliance with the known sequence anchors, namely the TM1TM2 loop and glycine-containing motifs.
pKa values of Glu61 were computed using the PROKA server
[51]. Figures were prepared using the PyMol molecular viewer
(http://www.pymol.org/).
Materials and Methods
CFoCF1 and c-ring purification
Thylakoid membranes were purified from ,800 g P. sativum
var. Alaska young leaves as in [39]. CFoCF1 complexes were
released from the membrane using 0.4% n-dodecyl-b-D-maltoside
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C-Ring Structure and Flexibility
middle, exhibiting tetradecameric symmetry [11] consisting of an
inner and an outer ring. The structure was 60.5Å long, with outer
ring diameters of 60.5Å and 61.5Å at the stroma and lumen sides,
respectively (Fig. 1A), whereas the inner ring had a diameter of
35Å at both sides of the membrane (Fig. 1B). The narrowest
diameter, 27Å, was mapped to the middle of the ring at Ile22 and
Gly23 (Fig. 1A). Each monomer was a hairpin-shaped, 81-residue
polypeptide, containing two trans-membrane (TM) helices; TM1
(residues 4–41) composed the inner ring, and TM2 (residues 46–
75) formed the outer ring. Both the C- and N-termini were
positioned towards the lumen. TM1 and TM2 were connected by
a short hydrophilic loop located at the stroma (residues 42–45).
TM1 and TM2 were both kinked towards the membrane (with
angles of 31u and 20u, respectively), with the kink mapped to the
region around Gly23 and Glu61.
According to the hydrophobicity profile [61], residues Leu54
and Phe76 line the hydrophobic boundaries of the c-ring,
surrounded by hydrophilic residues on both ends (Fig. 1C). This
indicates that the core membranal region of the c-ring spans 32Å,
which potentially correspond to the hydrophobic core width of
thylakoid membranes at the ATP synthase location. Additionally,
a pronounced hydrophilic patch is apparent at the center of the
membrane region, representing the proton-binding site, which is
further described below (Fig. 1C). Evolutionary conservation
analysis, mapped onto the c-ring structure, was in agreement with
the observed structural and functional features. The highly
conserved residues (receiving ConSurf grades of 8 or 9 [49])
included the hydrophilic membrane-exposed binding site and
surrounding residues, as well as the stroma-facing loop (Fig. 1D).
The latter is implicated in the interaction with the c-e globular
region, part of the F1 component [6]. Additional conserved
positions were detected at helix-helix interactions. Variable
positions, i.e., those receiving ConSurf grades of 1 or 2, mapped
to the remaining lipid-exposed residues (Fig. 1D), as well as to
positions facing the ring interior, a region that probably does not
possess a functional or structural role (Fig. 1D).
The proton-binding site retains a chemical coordination similar
to that of the alkalophilic cyanobacterium Spirulina platensis (Fig. 2),
including the same amino acids: Gln28 and Glu61, located on
TM1 and TM2, respectively, of the same monomer, and Phe59
and Tyr66, which are positioned on TM2 of an adjacent
monomer. All four residues were clearly identified in the electron
density map (Fig. S2). A network of hydrogen bonds stabilizes the
highly conserved Glu61 (Fig. 2).
Glu61 is likely to be protonated in the structure, which was
crystallized at pH 4.5, and this notion is supported by a pKa
analysis [51]. The Oe2 part of the carboxyl group of Glu61
generates hydrogen bonds with the Ne2 of Gln28 and the carbonyl
group of Phe59 (with distances between relevant atoms of 3.1Å
and 2.8Å, respectively). The second oxygen group, Oe1, forms a
hydrogen bond with the hydroxyl group of Tyr66 (2.9Å). This
chemical coordination represents a locked conformation of the
binding site [24].
GNM and ANM computations
In GNM and ANM, the structure is viewed as a collection of
nodes, derived from the Ca atoms, and springs, connecting the
nodes according to a given distance cutoff [36,37,46]. The normal
modes of motion are determined by the protein’s structural
architecture, and are ranked according to their 1/eigenvalues from
slow to fast, i.e. from the most cooperative modes to local
fluctuations. Based on the modes’ contribution to the overall
motion, derived from the 1/eigenvalues, we identified five main
modes of motion. As observed for other symmetric membrane
structures (e.g. [35,52,53,54,55]), some of the modes were
degenerative; two types of motion (types I and III, discussed
above) were each derived from a combination of two symmetryrelated, GNM modes, exhibiting essentially the same frequency.
We thus averaged the GNM fluctuations and inter-residue crosscorrelations of each pair of modes to receive symmetrical behavior
for all subunits. The three types of motion, referred to as types I, II
and III, consist of GNM1-2, GNM3 and GNM4-5, respectively.
The hinges were derived from the residue fluctuations of each type
of motion, denoted as minima, i.e., regions demonstrating
significantly low mobility relative to the rest of the residues. To
obtain the directions of these structural displacements in 3D space,
we employed ANM using the HingeProt [56] and the ANM
webservers [57]. The GNM modes were associated to the ANM
modes on the basis of inter-residue cross-correlations (Fig. S4).
The same analysis was performed for additional c-ring structures,
namely, Protein Data Bank (PDB) IDs 2x2v, 2xnd, 2xok, 1yce,
2w5j and 2wei [12,23,24,25,26,58]. Further details are available in
the Supporting Text S1.
Results
Overall structure of chloroplast ATP synthase subunit-c
The membrane-embedded domain of chloroplast ATP synthase, including subunit-c, was purified from the native holoenzyme by ion exchange chromatography, and further purified in
two precipitation steps with polyethylene glycol (See Materials and
Methods). This process, in contrast to previous pre-crystallization
processes for c-rings [59,60], was performed without any intense
treatment such as harsh detergent or heating. This approach
enabled us to crystallize the chloroplast c-ring of a higher plant (P.
sativum) from its native environment. The crystal structure of the
chloroplast ATP synthase c-ring was determined at 3.4Å
resolution with R-work/free of 29% and 32%, respectively, with
no outliers in the Ramachandran plot (Table 1).
The crystal lattice revealed one tetradecameric ring in the
asymmetric unit, forming crystal contacts with three additional
rings. One contact region consisted of loop-to-loop interactions
mediated by yttrium ions, as indicated by strong electron densities.
The remaining interactions involved the N-termini of three
monomers with three additional monomers of two symmetry
mates. This interaction was mediated by distinct electron density,
which was difficult to interpret (Fig. S1A). Interestingly, another
crystal contact was generated in the hydrophobic region located
between two rings (Fig. S1B). This inter-space was wide enough to
incorporate detergent molecules, which can replace the annular
layer of thylakoid membrane lipids or the lipids themselves.
Indeed, we observed electron density in this region that resembled
a lipid-like structure. The onset of the hydrophobic chain was
situated parallel to Phe76, mapped to the luminal boundary of the
thylakoid membrane, protruding towards the membrane center
(Fig. S1B).
The crystal structure of the c-ring from P. sativum (see Fig. 1)
shows a concave barrel shape with a pronounced waist in the
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Normal mode analysis
We analyzed the cooperative movement of the c-ring structure
in terms of elastic network models. The motion patterns of residues
and the cross-correlations between residues were computed using
GNM [36,37]. The directionality of this motion was derived from
associated ANM modes [38]. We focused on the five dominant
modes, which fell into three classes, referred to as types I, II and III
(Fig. 3A). Type I was two-fold degenerate, and the associated
deformation of the ring ellipsoidal. Type II was undegenerate and
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C-Ring Structure and Flexibility
Table 1. Data collection and refinement statistics.
to associate ANM modes to each type of GNM-derived motion
(Fig. S4).
Data statistics
Type I
Wavelength (Å)
Space group
C2
Unit cell parameters (Å,u)
a = 139.3, b = 102.54, c = 122.63
b = 101.22
Total reflections
79,289
Unique reflections
27,237
Completeness (%)
99.2 (86)*
Rpim (%)
14.5 (80)
I/s
In this type of motion, the inter-residue correlation separated
the c-ring into two oppositely-correlated dynamical elements,
divided by a plane perpendicular to the membrane (Figs. 4A and
4B). The associated ANM modes (ANM1–4; Fig. S4 and
Table S1) manifested an ellipsoidal deformation, emphasized at
the lumen-facing ends in ANM1 and ANM2, and at the stromafacing loops and helical regions in ANM3 and ANM4 (Figs. 4C,
4D and Movies S1 and S2). In this motion, the ring expands and
contracts; opposing monomers approach the center of the c-ring,
while the rest of the monomers simultaneously move outwards.
This motion changes the overall shape of the ring, transforming its
initial round shape into an elliptic one.
1.000 Å
3.3 (1.0)
6
6
Resolution range (Å)
82–3.4
X-ray source
ESRF beam line ID23-1
Type II
Refinement statistics
Resolution range (Å)
28–3.4 (3.58–3.4)6
No. of reflections (working/test)
20150/1044
dmin (Å)
3.4
Rwork/Rfree (%)
29/32 (0.39/0.45)6
In this motion, the c-ring structure was divided into two
dynamical elements, with a plane separating the helices into
lumen- and stroma- facing halves, passing through the hinge
region at the membrane center (Figs. 3B and 5A). One dynamical
element consisted of the helices’ lumen-facing halves (residues 3–
21 and 63–81), oppositely correlated to the second element, which
includes the stroma-facing halves and the short loop (residues 24–
60) (Fig. 5). Correspondingly, the matched ANM mode (ANM5;
Fig. S4 and Table S1) displayed a rotational motion of the two
dynamical elements in opposite directions (Fig. 5 and Movie S3),
referred to as a twisting motion. The lumen-facing halves rotated
clockwise while the stroma-facing halves simultaneously rotated
counter clockwise, and vice versa.
RMS deviation from ideality:
Bond lengths
0.008
Bond angles
1.2
Average B factor (Å2)
78
Solvent content (%)
63
*after anisotropic scaling.
highest resolution shell.
doi:10.1371/journal.pone.0043045.t001
6
Type III
the deformation torsional, and type III was two-fold degenerate
with kinks in the middle.
We carried out the same normal mode analysis for the
structures of six other c-rings of FoF1-ATPases, in which the
respective numbers of c-subunits varied from eight to fifteen, as
well as for the functionally-related ring of a V-ATPase, comprising
monomers of four TM helices each [27]. All revealed very similar
modes to those of the green pea c-ring, with the same three types
of motion (see Fig. S3, Table S1). The similarity of the dominant
slow modes among these different enzymes was not very surprising
considering their common toroidal topology. Comparable modes
have been reported for an unrelated toroid, the nuclear pore
complex [62].
In all three types of motion of the P. sativum c-ring, the hinge
regions, i.e. the least mobile residues in the GNM analysis, were in
similar locations. They were clustered at positions 21–26 and 60–
64, approximately at the kinked central regions of the two TM
helices (Fig. 3B and 3C). Notably, the essential acidic residue of the
proton-binding site, Glu61, was part of the hinge region. Gln28
and Phe59, also involved in the ion-binding site [24], resided in
close proximity to the hinge. The hinge region was more
pronounced, i.e. less mobile, in motion-types II and III, and was
less pronounced in type I (Fig. 3B). Although all three types of
motion shared the same hinge region, the inter-residue correlations significantly differed (Figs. 4A, 4B, 5, 6A and S4). Typically,
GNM and ANM modes are matched according to the residue
fluctuations, specifically, the location of the hinges. In this analysis,
however, as the hinge locations were essentially the same in all
classes of motion, they could not be utilized to correlate the GNM
and ANM results. We thus relied on the inter-residue correlations
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The inter-residue correlation divided the structure into four
main dynamical elements. Each of these four elements consisted of
either the lumen- or the stroma-facing halves of approximately five
monomers, divided by the hinges at the middle plane of the
membrane (Figs. 3B and 6A). Negative dynamical correlation was
detected between stroma- and lumen-facing halves of the same five
monomers (Fig. 6A). Positive dynamic correlation was observed
between stroma-facing halves and lumen-facing halves situated on
monomers of opposing sides of the ring (Fig. 6A). The matched
ANM modes (ANM6–9; Fig. S4 and Table S1) consisted of a
bending and stretching motion, governed by the hinges at the
center of the ring (Figs. 3B and 6B, Movie S4). The four dynamical
elements described above endured the largest structural displacements during the motion, while the monomers between them
mediated the motion. This bending and stretching motion altered
the exposure of the hinge region at TM2 towards the membrane.
Discussion
This work presents a hybrid approach of X-ray crystallography
and computational analysis to reveal both fine and gross structural
properties of the c-ring of the FoF1-ATPase in higher plant
chloroplasts. We discuss the three types of elastic slow modes and
associate them with the rotary ion translocating function of the cring, in interaction with subunits a and bb’.
Crystal structure of the c14-ring of the FOF1-ATPase in the
pea
High-resolution crystal structures of the FoF1-ATPase c-rings of
various organisms have been determined [6,12,23,24,25,26]. The
ring’s resilience to high temperature and harsh detergents has
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C-Ring Structure and Flexibility
Figure 1. Architecture of the chloroplast ATP synthase subunit-c. A&B Side (A) and stroma (B) views of the ring, with differently colored
monomers. The ring dimensions are marked, and blue arrows indicate the narrowest ring region. C. Side view of the structure in surface presentation,
colored according to the hydrophobicity scale below. Left: the membrane boundaries according to the hydrophobicity profile are marked, mapped
to Leu54 and Phe76. The hydrophilic residues reside at the ring edges, corresponding to extra-membrane regions, as well as at the membrane center,
at the proton-binding site. Right: slab view, displaying the interior of the ring. D. The structure is viewed as in panel C and colored according to
evolutionary conservation as calculated by the ConSurf webserver (http://consurf.tau.ac.il, [49]), with cyan-to-maroon indicating variable-toconserved positions, according to the color bar. Left: the hydrophilic proton-binding site at the membrane center is highly conserved, as are the
stroma-facing loops. Right: a slab view reveals that residues lining the interior of the ring are highly variable. Indeed, this region is not expected to
possess a functional or structural role.
doi:10.1371/journal.pone.0043045.g001
facilitated the preparation of 2D-crystals for atomic force
microscopy and electron microscopy and of 3D-crystals for Xray structure analysis. It is an interesting possibility that
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crystallization under harsh conditions ‘‘purifies’’ certain structural
variants. To address this, we used a very mild preparation and
crystallization technique, starting from the native holoenzyme. In
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C-Ring Structure and Flexibility
Figure 2. Structural alignment of the c-ring proton-binding
site. Structural alignment of the crystal structure of the green pea
subunit-c (colored green) and the crystal structure of the protonated
state of the cyanobacterium c-ring (PDB ID 2wie, colored yellow). The
proton-binding site of the two structures as viewed from the
membrane plane is shown, with the lumen below. The residues of
the binding site are marked, and the bond length is shown in Å. The
alignment indicates that the structural arrangement and chemical
coordination of the proton-binding site are conserved between
cyanobacteria and higher plants. It should be noted that according to
the crystal structure of the spinach c-ring [12], the chemical
coordination of the binding site differs from that of the binding sites
of the other c-rings shown herein. Yet, MD simulations indicate that
chemical interactions similar to those observed for the cyanobacteria
and the green pea binding sites are more stable in the spinach site as
well [29].
doi:10.1371/journal.pone.0043045.g002
order to overcome the common heterogeneity of plant material, P.
sativum var. Alaska was used under controlled growth conditions.
By utilizing a mild approach, we hoped to capture a native form of
the c-ring structure, revealing the native protein/lipid-interaction.
To the best of our knowledge, the special electron density detected
at the crystal contacts between hydrophobic regions of the c-ring is
a unique feature, which has not been observed to date in other
crystal structures of membrane proteins (Fig. S1B). A galactolipid
was tentatively modeled into the intercalating electron density, in
compliance with the notion that the uncharged heads of the
galactolipids enable their penetration through the thylakoid
membrane, as was demonstrated in the crystal structure of
cyanobacterial photosystem II [63]. We expect that our preparation and crystallization approach would be useful for future
investigations aimed at exploring the effect of lipids on the
functionality of membrane proteins. Moreover, it could be highly
effective for studying and determining the structure of the a-c
complex of the FoF1-ATPase.
Despite the high sequence identity between the c subunit in the
green pea and the c subunits of other photosynthetic organisms
(e.g., subunit-c of spinach spp. [12] and S. platensis [24], both
displaying more than 85% identity to the green pea subunit-c),
there are distinguishable structural differences among them. The
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Figure 3. The three classes of motion, derived from GNM:
contribution to overall motion, fluctuations and hinges. A.
Contribution of the 30 slowest GNM modes to the overall motion. The
contribution was calculated based on the 1/eigenvalues values of the
modes, derived from the GNM calculation. As the mode pairs GNM1-2
and GNM4-5 display the same contribution, i.e. the same eigenvalues,
they are considered degenerate modes, with the actual motion
consisting of an average of each pair. Overall, there are three main
types of motion, denoted as types I, II, III, corresponding to GNM1-2,
GNM3 and GNM4–5, respectively. B. The mean square fluctuations
(MSFs) are shown for one monomer, with the derived hinge regions
shaded in yellow. The insert in the upper left shows the MSFs for all
monomers, whereas the insert in the upper right displays the c-ring
structure from a side view, with the hinge residues as yellow spheres. It
is evident that while all three types of motion share the same hinge
regions, these are prominent only in motion types II and III and not in
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September 2012 | Volume 7 | Issue 9 | e43045
C-Ring Structure and Flexibility
electron density map [12]. Recent MD simulations indicate,
however, that the original coordination of the proton-binding site
is unstable, and therefore rapidly incorporates the bacterial-like
coordinates [29]. In sum, the FoF1-ATPase c subunits derived
from the chloroplasts of higher plants and from their ancestors, the
oxygenic photosynthetic bacteria, share a chemical coordination
and a hydrogen bond network that stabilize proton binding by the
essential glutamate on subunit c.
type I. C. Sequence alignment of c-rings with available structures. The
yellow marking indicate the hinge region of types II and III in the GNM
analysis of the green pea c-ring. In all rings, the hinge region was
assigned to overlapping regions, with the second hinge including the
functional glutamate. The alignment displays the region corresponding
to positions 9 to 76 of the green pea subunit-c, omitting parts of the Nand C-termini.
doi:10.1371/journal.pone.0043045.g003
Network model to assess the dominant elastic modes of
the c-ring
proportions of the P. sativum c-ring (height/width 60.5Å/60.5Å)
differ slightly from those of its spinach homologue (65Å/58Å,
respectively [12]), while its cyanobacterial homologue, with 15
monomers on the ring, is similar (65Å/65Å, [24]). Similarly, the
coordination pattern at the proton-binding site in the pea c-ring
resembles that in the cyanobacterium (Fig. 2) yet differs from that
of the spinach homologue. In the latter structure, additional
hydrogen bonding was detected between the hydroxyl groups of
Thr64 with Oe1 of Glu6, and Gln28 was not detected in the
The elastic network computational approach we used was based
on the coarse-grained topology of the protein. It accounts only for
the Ca-atoms and yields the eigen-modes of an undamped elastic
network. In several cases it has been demonstrated that the few
dominant (slow) eigen-modes observed for a given protein can be
associated with large-scale and long-range functional movements
that are pivotal for that protein’s mechanism [33,35]. In a real
protein that is embedded in a solvent (the viscous membrane in
Figure 4. Motion Type I. A. Dynamical correlation between all residues in motion type I, derived from GNM. The correlation values range from blue
to red, indicating negative and positive dynamical correlation, respectively, according to the scale. The different chains are marked on the matrix. B.
The GNM dynamical correlation is mapped onto the c-ring. The c-ring is shown in cartoon representation and is viewed from the stroma. C & D. The
ANM modes corresponding to motion type I. ANM1 (panel C) and ANM3 (panel D) are shown as cartoons and viewed from the lumen and stroma,
respectively. The deformations are colored from gray to blue; arrows indicate the direction of motion and dotted circles mark the extreme
deformations. In both modes, the c-ring expands and contracts, with oppositely correlated monomers moving towards the ring center, while the rest
of the monomers move outwards. This results in an elliptic conformation.
doi:10.1371/journal.pone.0043045.g004
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C-Ring Structure and Flexibility
dominant global modes of the c-ring with its rotary function in
FOF1 and suggest a possible functional role.
Motion of type I
The contraction and expansion of the ring deform the ring
elliptically (Figs. 4C and 4D, Movies S1 and S2). Expansion of the
ring towards the a-subunit could favor closer apposition of the ion
binding pocket on subunit-c towards the essential arginine on
subunit-a. This might strengthen the electrostatic interactions
between the unprotonated Glu61 and the arginine (Fig. 7A, right).
On the other hand, the apposition of the flat face of the ring might
facilitate the contact of Glu61 for protonation/deprotonation
through either of the proton half-channels (Fig. 7A. left). The
deprotonation of one Glu through one half-channel together with
the protonation of the previously deprotonated Glu on the
adjacent monomer on the ring imply changing the c-subunit copy
that interacts electrostatically with the essential arginine on subunit
a. In other words, this motion advances the rotation of the c-ring
by one step.
Figure 5. Motion Type II. A. The deformations of motion type II
(associated with ANM5), colored according to dynamical correlation
from GNM. The c-ring is shown in cartoon representation and viewed
from the side, with the lumen below. The two dynamical elements
identified for this motion-the stroma-facing half and the lumen-facing
half, separated by the hinge region at the center of the ring are shaded,
respectively, in blue and in red. The deformations, colored from whiteto-red and from white-to-blue, show opposite rotational twisting of the
two dynamical elements, as marked by the arrows. B&C Stroma-facing
(B) and lumen-facing (C) views of the motion as shown in panel A, with
the arrows indicating the direction of motion.
doi:10.1371/journal.pone.0043045.g005
Motion of type II
Motion of type II twists the stroma- and the lumen-facing
surfaces against each other around the ring’s axis (Figs. 5 and 7B,
Movie S3). It is conceivable that this type of deformation
contributes to the previously established elastic buffer between
Fo and F1 [17,19,20,21]. The latter studies do not establish the
extent to which subunits c or e of F1 or the c-ring contributes to
the high elastic compliance of this buffer. Based on the present
analysis, we propose that the twisting deformation of the c-ring
(Fig. 5) is part of the elastic buffer between ATP synthesis and
proton transport (Fig. 7B).
this case), the elastic vibrations of each mode would be overdamped. Instead of oscillating, the domains stochastically fluctuate
in the elastic potential well associated with a given mode.
The normal mode analysis in the present work was restricted to
the isolated c-ring of the green pea chloroplast. The c-ring’s
interactions with both subunits a and bb’ in the membrane, and
with c and e of the F1-portion, were neglected. We assumed that
these intrinsic classes of motion of the c-ring are maintained within
the entire c14abb’-complex. This assumption is supported by the
literature, which has shown that isolated units intrinsically display
the motion corresponding to that of the physiologically-relevant
complex. GNM fluctuations of isolated monomers and those
derived from a complete complex have been successfully employed
to assess docking models [30].
We applied the same network analysis to c-ring structures from
other organisms, varying in size, shape and the number of
monomers in the c-ring (Figs. 3C and S4, Table S1). These
analyses yielded the same patterns of slowest modes. Similar results
have been obtained for other, unrelated toroid structures, for
instance the nuclear pore complex [62].
Motion of type III
Motion of type III bends and stretches the structure as
displayed in Figures 6B and 7C (see Movie S4). This motion is
expected to affect the exposure of Glu61, situated at the hinge
point. Upon proton release and before binding to subunit-a,
negatively charged Glu61 could possibly become exposed to the
membrane environment, which is thermodynamically unfavorable. The bending of the helices around the hinge position
(Fig. 6B and Movie S4) could thus partially shield the charged
acidic residue from the hydrophobic lipids, stabilizing this
intermediate state. We suggest that Ala58, Ile65 and Tyr66
might play key roles in this process, as these are highly conserved
residues around Glu61 according to our conservation analysis
(Fig. 1C). Specifically, hydrophilic Tyr66 might change its
original interaction with Glu61 (Fig. 2), masking it from the
hydrophobic environment via its bulky ring (Fig. 7C). Interestingly, when simulating a deprotonated state of the glutamate,
Pogoryelov et al. observed that the helix carrying the deprotonated glutamate is more strongly kinked [64], which is
compatible with the present analysis. Our results, however,
cannot account for straightening of an adjacent helix encompassing a protonated glutamate, also demonstrated by the above
researchers’ simulations. The stretching of the helices, on the
other hand, could increase the exposure of Glu61 towards the
membrane. This, in turn, could position the deprotonated Glu61
closer to the essential positive charge on subunit-a (Figs. 6B and
7C).
Functional interpretation of the three types of motion
The mechanism of rotary proton translocation and torque
generation relies on the interaction between the ring of c-subunits
and subunits a and bb’. The interplay between firm attachment
and rotary fluctuations of the c-ring and subunits a and bb’ has
remained to be characterized in detail. It has not yet been
determined whether this interplay is mainly governed by (i) local
conformational fluctuations (e.g., of residues in the ion-binding
pocket of subunit c [64], or of the five helices of subunit a), or (ii)
more global movements of the c-ring, as emphasized in this work.
In the following, we tentatively correlate the three classes of
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Conclusion
Elastic network analysis of the cn-ring of the FoF1-ATPase
demonstrated five dominant modes of motion, and we interpreted
8
September 2012 | Volume 7 | Issue 9 | e43045
C-Ring Structure and Flexibility
Figure 6. Motion Type III. A. The c-ring structure is colored according to the GNM-derived dynamical correlation, with positive-to-negative
correlation colored according to the red-to-blue scale. Four main dynamical elements are identified, mapped to the lumen- and stroma-facing halves
of the monomers at opposing sides of the ring (marked by red and blue shading). It is apparent that the stroma-facing halves are negatively
correlated with their lumen-facing halves, while positively correlated with the lumen-facing halves of monomers situated at the opposing side of the
ring. The left-hand side shows a side view, while the right-hand side displays stroma and lumen views. B. The deformations of the corresponding
ANM motion (ANM6). The structure is colored according to the correlation of the four main dynamical elements identified in panel A. Left: ANM
deformations, ranging from white to red or from white to blue, with the direction of motion marked. Right: the two extreme deformations,
corresponding to the two potential directions of this type of motion, with the main dynamical elements colored red or blue according to their
correlation. These deformations describe a bending and stretching motion originating from the hinge at the ring center, with bending of the helices
occurring at one side of the ring, while stretching takes place at the opposing side.
doi:10.1371/journal.pone.0043045.g006
overdamped, and the harmonic dynamics are converted into
stochastic fluctuations. We suppose that the relaxation times are
shorter than the transit time of proton transfer, which is
approximately 1024s (at 200 mV driving force) [31]. Although
the conformational fluctuations may facilitate rotary proton
transfer and torque generation by Fo, they do not limit the rate,
as evident from the ohmic character of proton conduction by Fo
[31].
the roles of these modes in the interaction of the c-ring with its
counterpart in Fo (the a and bb’ subunits) and with subunits c and
e of F1. Thus, these modes affect (i) the dynamics of sliding versus
binding of cn relative to abb’, (ii) proton transfer between c and a,
and across the membrane, and (iii) the elastic torque transmission
between Fo and F1. The coarse-grained elastic network formalism
yields global modes of elastic vibrations. For a protein that is
embedded in a solvent (here the membrane), these vibrations are
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C-Ring Structure and Flexibility
Figure 7. Cartoon illustrating the functional interpretation of the dominant modes. Schematic presentation of the c-ring (light green),
subunit-a (dark gray) and the c-e complex (pink). Protonated Glu61 of the c-ring, the conserved Arg of subunit-a and Tyr66 are shown as gray, blue
and green asterisks, respectively, with unprotonated glutamates as red asterisks. A. Motion type I. Top view. Right: Upon ring expansion, the
electrostatic interaction between Glu61 and the Arg is strengthened, while the probability of proton transfer is weakened. Left: Upon contraction, cring places a flat face toward subunit-a. This interaction could facilitate protonation/deprotonation of Glu61 through the two proton half-channels on
subunit-a (dashed black arrows). It results in one step of counter-clockwise rotation (green arrows). The cartoon suggests three sites of interaction
between subunits c and a, whereas it has been suggested that the Arg on subunit-a has two functions [65], resulting in two interaction sites. This
alternative does not impair the above interpretation regarding effect of this subunit-c deformation. B. Motion type II. Side view with the thylakoid
lumen below and the stroma side above. Helices are illustrated as gray cylinders, with the lumen-facing halves in lighter shade. As the c-e subcomplex undergoes a-120u rotation (marked by pink arrows) and the c-ring rotates by a smaller step [21], the twisting motion of the stroma-facing
helical halves (colored dark gray) is suggested to serve as an elastic buffer for smooth torque transfer between FO and F1 (marked in directions 1 and
2 by black arrows). The opposite rotational directions illustrate the two potential functionalities, i.e. hydrolysis and synthesis. C. Motion type III.
Same view as panel B, with TM2 illustrated (light gray). 1. As the structure stretches towards the left side, Glu61 of the leftmost subunit becomes
more exposed to the membrane, which could enhance its interaction with the Arg of subunit-a. 2. Bending of the structure masks Glu61 from the
membrane, with potential contribution of Tyr66. Again, this motion could facilitate proton exchange between the two residues.
doi:10.1371/journal.pone.0043045.g007
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C-Ring Structure and Flexibility
Comparison between normal mode analyses
of different c-rings. For each ring [9,10,11,12,13,14,15], the
GNM and ANM modes corresponding to motions of types I, II
and III are specified (colored green, blue and red, respectively).
Modes were matched according to the ANM deformations as well
as the GNM cross-correlations, as exemplified in Figure S4.
(DOCX)
Table S1
Supporting Information
Figure S1 Crystal contacts and packing. The c-ring is
viewed in cartoon representation. A. Crystal contacts between the
c-ring (blue) and its adjacent symmetry mates (green), with
interactions of either the stroma loops or the N- and C- termini. B.
Crystal contacts at the hydrophobic region between adjacent rings,
mediated by density corresponding to single lipid molecule. A digalactolipid is modeled within the lipid density. The hydrophobic
moiety of the lipid is situated exactly at the predicted hydrophobic
core of the membrane (Fig. 1C). Simulated annealing omit map
(Fo–Fc) contoured at 1s.
(TIF)
Movie S1 Motion type I – ANM1. The deformations of
ANM1, matched to the first type of motion, are shown as cartoons
and viewed from the lumen. The coloring corresponds to the interresidue correlation of chain A of GNM1-2, ranging from red to
blue (Figs. 4A and 4B). The motion manifests extraction/
contraction of the ring emphasized at the lumen end, with
oppositely correlated dynamical elements from the two sides of the
ring simultaneously approach the ring center and move away from
it.
(MOV)
Figure S2 Electron density map of the proton binding
site. Side view of the c-ring, with the lumen below. The binding
site residues are shown as sticks, with their attributed electron
density shown as mesh. Density map (2Fo–Fc), contoured at 1.3s.
(TIF)
Movie S2 Motion type I – ANM3. Same as Movie S1, with
ANM3 viewed from the stroma. This shows the extraction/
contraction motion manifested at the stroma-facing ends.
(MOV)
Figure S3 Comparison of the slowest types of motion of
the green pea c-ring, c-ring of Bacillus pseudofirmus
(PDB ID 262v) and c-ring of the bovine F1-c8 subcomplex (PDB ID 2xnd). The deformations of the corresponding types of motion (Table S1) are shown, colored by their GNMderived cross-correlations, according to the color bar below, with
negative (blue) to positive (red) correlation ranges between -1 and
1. Arrows indicate the direction of motion. This comparison shows
that although the rings are of different sizes and shapes, their three
slowest types of motion correspond to each other. Similar results
were obtained for the rest of the rings (Table S1). Note that for
PDB ID 2xnd, the order of modes differs from that of the green
pea c-ring, although the types of motion are the same (Table S1).
(TIF)
Movie S3 Motion type II – ANM5. The ANM5 deformations
are viewed as cartoons from the side, with the lumen below. The
colors, corresponding to GNM3 (Fig. 5), depict two oppositely
correlated dynamical elements, consisting of the lumen- and
stroma-facing halves, separated by the hinge at the ring center.
The two dynamical elements undergo twisting motions in opposite
directions.
(MOV)
Movie S4 Motion type III – ANM7. The deformations
predicted by ANM7 are shown from the side and colored by the
cross-correlations of the N-terminal of chain A with the rest of the
structure, blue-to-red indicating negative-to-positive correlation.
The motion consists of bending and stretching of the structure
towards the membrane, affecting the exposure of the hinge region,
including Glu61.
(MOV)
Association of GNM and ANM modes. As all
types of motion consisted of the same hinge regions (Fig. 3A), we
matched the GNM and ANM modes using their inter-residue
cross-correlations. For motion type I, derived from the average
GNM1-2 mode, ANM1 displayed a very similar cross-correlation
distribution. The exact same matrix was observed for ANM2,
ANM3 and ANM4 (data not shown), indicating that these modes
correspond to GNM1-2 as well. The inter-residue correlation of
GNM3 (motion type II) was matched to ANM5, whereas GNM45, representing motion type III, was associated with ANM6, as
well ANM7, ANM8 and ANM9, manifesting the same motion.
(TIF)
Figure S4
Acknowledgments
We thank Professor Turkan Haliloglu for helpful discussions.
Author Contributions
Conceived and designed the experiments: SS MS WJ NN. Performed the
experiments: SS MS. Analyzed the data: SS MS WJ NN. Contributed
reagents/materials/analysis tools: NBT NN. Wrote the paper: SS MS
NBT WJ NN.
Text S1 Normal mode analysis: GNM and ANM.
(DOCX)
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September 2012 | Volume 7 | Issue 9 | e43045