Molecular Plant • Volume 7 • Number 5 • Pages 916–919 • May 2014
LETTER TO THE EDITOR
Crystal Structure of a Multilayer Packed Major
Light-Harvesting Complex: Implications for Grana
Stacking in Higher Plants
Dear Editor,
Grana thylakoids in plants comprise multiple, tightly
appressed thylakoid membranes in the chloroplast, which
greatly increase the area-to-volume ratio and significantly
improve the ability of chloroplasts to capture light. Grana
layers are stacked and interact through the stromal surface of
proteins embedded in the grana membranes, mainly photosystem (PS) II components, including the core complexes, the
minor light-harvesting complexes (Lhcb4, b5, and b6) as well
as the major light-harvesting complex (LHCII). Among them,
LHCII, which is the most abundant membrane protein on Earth
and accounts for approximately one-third of the total protein
in plant thylakoids, is reported to play an essential role in
grana stacking. In the chloroplast of Arabidopsis knockout
mutant gdc1-3, which lacks trimeric LHCII, stromal thylakoids
could not stack together to form appressed grana (Cui et al.,
2011). Reconstitution of isolated purified LHCII into native
membranes lacking the complex restores the ability to stack
under physiological conditions (Day et al., 1984). In addition,
membrane stacking was also observed in liposomes reconstituted with LHCII in the presence of cations (McDonnel and
Staehelin, 1980). Taken together, these earlier insights provided evidence that trimeric LHCII has a central role in grana
adhesion. Intriguingly, according to the previously reported
crystal structures, the stromal surface of LHCII is essentially
negatively charged. Moreover, the innate lipid components
of thylakoids, SQDG and PG, also carry negatively charged
chemical groups (sulfate and phosphate groups, respectively).
Then the question is raised: how do the negatively charged
thylakoids stack together?
The spinach chloroplast contains approximately 18 mM
Mg2+ and 200 mM monovalent cations (mainly Na+ and K+)
under normal growth conditions (Schroppel-Meier and Kaiser,
1988). It is now well established that thylakoid membrane
stacking is reversibly lost by suspending isolated thylakoids in
‘low-salt’ solutions (≤20 mM monovalent cations). Re-addition
of low concentrations of divalent cations (≥5 mM Mg2+) or
higher concentrations of monovalent cations (≥150 mM Na+
or K+) restores grana stacks in these low-salt thylakoids (Izawa
and Good, 1966). Mild trypsin digestion of the intact thylakoid
membrane removes the N-terminal segment of LHCII, which
significantly increases the cation concentration required for
thylakoid membrane restacking from 5 mM Mg2+ to 15 mM
Mg2+ (Carter and Staehelin, 1980). These observations show
that both cations and the N-terminal segment of LHCII play
important functions in grana stacking, yet their specific roles
remain elusive.
Based on the crystal-packing pattern of pea LHCII (PDB
entry 2BHW), a ‘Velcro’-like model of grana stacking was proposed, implying the primary attraction force between adjacent layers is the non-specific columbic interaction between
the positively charged N-terminal residues and the negatively
charged stromal surface of LHCII (Standfuss et al., 2005).
While this view explains the importance of LHCII N-terminal
in grana stacking, it seems incomprehensive with the fact
that, when the N-terminal segment of LHCII is removed, the
thylakoid membrane can still stack at higher salt conditions
(Carter and Staehelin, 1980), which is still within the physiological range. Besides, the LHCII trimers in the crystals of
2BHW adopt an up-and-down arrangement in the membrane
plane that avoids the face-to-face contacts between negatively charged stromal surfaces (Figure 1A–1C). Apparently,
this is not the situation in the grana membranes under in vivo
conditions (Daum et al., 2010).
To better address the role of LHCII in mediating grana
stacking, we crystallized the spinach LHCII under physiological-relevant cation strength (10~30 mM Zn2+, 200 mM Na+,
and pH 6.5) in the presence of 2 mg ml–1 native lipids, DGDG,
and solved the structure at 2.6 Å resolution (Supplemental
Table 1). The protein sample used in our crystal preparation
was trypsin-digested, thus it did not contain the positively
charged N-terminal. Despite that the V1 site for violaxanthin binding is vacant, there are no other significant differences between the present structure and the two previously
reported structures (supplemental results). However, the
packing patterns of these crystals are fundamentally different. The new crystal form contains multilayer stacks of LHCII
trimers which are unidirectionally inserted into the membrane plane (Figure 1E and 1F). Over half of the stromal surfaces of LHCII are packed face to face (Figure 1G), forming
extensive contacts between chain B and chain C with their
© The Author 2014. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPB and
IPPE, SIBS, CAS.
doi:10.1093/mp/ssu005, Advance Access publication 30 January 2014
Received 27 October 2013; accepted 9 January 2014
Letter to the Editor 917
Figure 1. A Structure-Based Model of Grana Stacking.
(A–C) Crystal packing in pea LHCII crystals (PDB entry 2BHW).
(D) The physiological stacking pattern of LHCII in grana revealed by electron tomography. The red and green masks shown in (C), (D), and (G) were
adapted from the electron tomography study reported by Daum et al. (2010) , and the material is copyrighted by the American Society of Plant
Biologists and is reprinted with permission.
(E–G) Crystal packing in our present structure (PDB entry 4LCZ). (A, E): side view of the crystal packing of 2BHW and 4LCZ, respectively. (B, F):
schematic representation of the crystal packing of 2BHW and 4LCZ, respectively. In 2BHW, the LHCII trimers are packed in an up-and-down manner
and formed limited stromal contacts, while in 4LCZ, a large area of face-to-face stromal contacts was observed. (C, G) Roughly fitting of the crystal
packing of 2BHW and 4LCZ into the in situ stacking of LHCII illustrated in (D). It was demonstrated that the packing pattern of 4LCZ can be well
fitted into the LHCII stacking pattern in grana observed through electron microscopy (G).
(H) Surface charge representation illustrated the negatively charged stromal surface of LHCII. Positively charged regions are shown as blue and
negatively charged regions are shown as red. Cations were located in negatively charged regions to screen the net negative charges.
(I) Distribution of flexible grana-stacking residues across the stromal surface of LHCII. The residues distribute evenly across the surface, making it
brush-like.
(J) Illustration of the interactions between stromal residues and cations in neighboring layers in the crystal.
(K) A model of grana stacking in grana based on our crystal packing. The negatively charged stromal residues are represented by red sticks. Cations
are shown by blue spheres. The positively charged N-terminals are represented in blue. This model highlights the roles of both cations and the
N-termini in grana membrane stacking. The salt bridges are illustrated to be the primary attraction forces in stacking.
918 Letter to the Editor
symmetry mates, closely resembling the interlayer overlapping pattern of LHCII revealed by recent electron topographical examination on PSII and LHCII super-complexes in isolated
grana thylakoids (Daum et al., 2010) (Figure 1G). Thus, the
pattern of our crystal packing provides a possible simulation
of LHCII stacking in grana membranes in vivo and it may serve
as an improved model in characterizing the attraction forces
between adjacent grana layers.
Eight zinc ions and three cacodylate ions were located
for each trimer by the anomalous difference Fourier maps
(Supplemental Figure 1). In addition, five strong and spherical densities were assigned as sodium ions (Supplemental
Figure 2; for cation identification, see supplemental results).
Among all these cations, three Zn2+ ions partially replace the
central Mg2+ of Chl a614 in each monomer (Supplemental
Figure 1A), indicating the flexible and solvent-accessible
nature of Chl a614 in the structure presented here. Cations
on the stromal surface, including five Na+ ions (Na1, Na1′,
Na2, Na3, and Na3′) and two Zn2+ ions (Zn1 and Zn1′) contribute significantly to the stromal packing of the crystal
(the symbol ‘ here represents the symmetry-related molecule) (Figure 1H), which bridge negatively charged residue
pairs from neighboring layers through strong ionic bonds
(Supplemental Table 2), constituting the primary attraction force between neighboring layers. Na1 and the central Zn2+ ion mediate the interactions between Glu31/B′
and the three Asp54 residues in the trimer (Supplemental
Figure 2A). Na2 locates exactly on the crystallographic
two-fold axis and bridges the interactions of Glu171/C~
Glu171/C′ (Supplemental Figure 2B). Na3 is involved in the
interplay of the residue cluster Ser34/A~ Asp20/B ′~ Glu56/B
(Supplemental Figure 2C). Several hydrogen bonds, to a
lesser extent, also contribute to the stabilization of crystal
packing in the stromal surface (Supplemental Table 3). Based
on the multiple cation binding sites and the large stromal
contact area, we suggest that the structure described here
probably represents a specific mode of LHCII stacking in
grana, and thus provides a reasonable explanation on how
cations contribute to the stacking of the grana membranes
in vivo.
Nine stromal-exposed residues (shown in Figure 1H), which
are responsible for either the coordination of cations or the
formation of hydrogen bonds between neighboring layers, are conserved across species (Supplemental Figure 3),
whereas only five residues in another PSII antenna protein,
Lhcb5, are conserved in Arabidopsis when aligned to Lhcb1.
The Arabidopsis antisense plant asLhcb2, which contains
Lhcb5 trimers in the thylakoids, can also form a grana ultrastructure (Andersson et al., 2003), but is less stable than wildtype plants. This evidence suggest that these residues play
important roles in grana stacking. Except for Ser34 and Thr37,
all the remaining residues are negatively charged. The side
chains of most of the residues have significantly higher average B factors comparing to the overall B factors of the protein
in all three available structures (Supplemental Table 4) and,
when the different monomers from the asymmetrical unit
in the reported structures were superposed, different sidechain conformations were observed, demonstrating their
flexible nature. These residues are evenly distributed across
the LHCII stromal surface, rendering the entire surface flexible (Figure 1I).
Based on the newly observed crystal-packing pattern,
we estimated that the distance between stromal surfaces
of neighboring grana stacks is about 22 Å (Supplemental
Figure 8), which is approximately in line with the previous
report of about 2-nm partition gap (Dekker and Boekema,
2005). Within this range of distance, the grana membranes
may easily stack through specific protein–protein interactions
between adjacent stacks and thus form ordered semi-crystalline arrays of PSII–LHCII super-complexes observed in native
grana membranes. Besides, a slightly larger partition gap may
also exist in grana stacks, maybe in different regions or under
different light conditions (supplemental results).
According to the structural analysis described above, in
combination with previous biochemical data, a model of
grana stacking was proposed (Figure 1K). In this model, LHCII
molecules are stacked face to face between adjacent membrane layers. Cations are sandwiched between the neighboring layers to screen the surface charge and mediate the
interactions by forming strong salt bridges, making them the
primary stabilizing force in grana stacking. The interactions
between the stromal residues and cations may have fairly
dynamic behavior due to the flexibility of these surface residues, which are probably important for protein trafficking
within the grana membrane and for the elaborate dynamic
change of the grana ultrastructure in response to changing
light conditions. It is noteworthy that, within this model,
the flexible N-terminal segments of LHCII can still protrude
out to interact with the negatively charged stromal surface
outside the overlapping interface, thus contributing to the
overall stability of grana ultrastructure in plant thylakoids
(Figure 1K).
Grana stacking probably involves all the components of
PSII, including the peripheral antenna and the core subunits.
Previous reports indicated that other PSII components, such
as the core complexes, also play important roles in grana formation with a similar cation-dependent manner (Burke et al.,
1979), suggesting cations can be bound to the stromal surface of these PSII components, possibly in a similar manner as
in LHCII. Thus, our model provides a starting point in understanding the molecular interplays between grana stacks, and
cations are most likely a general mediating component in
bridging the protein–protein interactions between opposing
membrane stacks in grana.
SUPPLEMENTARY DATA
Supplementary Data are available at Molecular Plant Online.
Letter to the Editor FUNDING
This work was supported by the 973 Project (Grant No.
2011CBA00902) and the National Natural Science Foundation
of China (Grant No. 31021062 and 31270793).
Acknowledgments
We are grateful to the staff of beamline 17U1 at the Shanghai
Synchrotron Radiation Facility and to the staff of the Photon
factory and Spring-8 Synchrotron Radiation Facility for excellent technical assistance during data collection. We thank Dr.
Hongmei Zhang for technical support during structure refinement and Prof. Fei Sun for assistance in the interpretation of
the electron tomography data in the reference cited in the
manuscript. No conflict of interest declared.
Tao Wana,b, Mei Lia,1, Xuelin Zhaoa, Jiping Zhanga,
Zhenfeng Liua, and Wenrui Changa,1
a National Laboratory of Biomacromolecules, Institute of Biophysics,
Chinese Academy of Sciences, 15th Datun Road, Chaoyang District, Beijing,
100101, People’s Republic of China
b University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing,
100049, People’s Republic of China
1
To whom correspondence should be addressed. M.L. E-mail meili@moon.
ibp.ac.cn, tel. 86-10-64888507, fax 86-10-64889867. W.C. E-mail wrchang@
sun5.ibp.ac.cn, tel. 86-10-64888512, fax 86-10-64888512.
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