Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex

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Science  25 Aug 2017:
Vol. 357, Issue 6353, pp. 815-820
DOI: 10.1126/science.aan0327

Adapting to the right light

In plants, photosystem II is the first protein complex in the machinery that converts sunlight into chemical energy. It comprises antennae complexes (LHCIIs), which collect the light energy, and a dimeric core that contains the reaction center where water is split into oxygen and protons. Su et al. report cryo-electron microscopy structures of a supercomplex consisting of the dimeric core, two strongly bound LHCIIs, and two moderately bound LHCIIs (see the Perspective by Croce and van Amerongen). Under high-light conditions, the moderately bound LHCIIs might detach to down-regulate the efficiency of light harvesting and prevent damage.

Science, this issue p. 815; see also p. 752


In plants, the photosynthetic machinery photosystem II (PSII) consists of a core complex associated with variable numbers of light-harvesting complexes II (LHCIIs). The supercomplex, comprising a dimeric core and two strongly bound and two moderately bound LHCIIs (C2S2M2), is the dominant form in plants acclimated to limited light. Here we report cryo–electron microscopy structures of two forms of C2S2M2 (termed stacked and unstacked) from Pisum sativum at 2.7- and 3.2-angstrom resolution, respectively. In each C2S2M2, the moderately bound LHCII assembles specifically with a peripheral antenna complex CP24-CP29 heterodimer and the strongly bound LHCII, to establish a pigment network that facilitates light harvesting at the periphery and energy transfer into the core. The high mobility of peripheral antennae, including the moderately bound LHCII and CP24, provides insights into functional regulation of plant PSII.

Photosynthesis carries out a series of fundamental biophysical and biochemical processes to convert solar energy into chemical energy. Oxygenic photosynthetic organisms split water molecules into oxygen and protons through the molecular machine photosystem II (PSII) that is embedded in the thylakoid membrane and consists of a dimeric core associated with peripheral antenna complexes (1, 2). The peripheral antennae collect light energy and transfer it to the reaction center, where the water-splitting reaction is catalyzed by the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) (3). In plants, the PSII core complex comprises at least 20 subunits. As shown in Fig. 1A, most of them are membrane-intrinsic proteins, including the reaction center subunits (D1, D2), inner antennae (CP43, CP47), and numerous low–molecular mass subunits including cytochrome b559 (a heme-bridged heterodimer composed of PsbE and PsbF). In addition, it binds several extrinsic subunits on the luminal side, including OEC proteins (PsbO, PsbP, PsbQ) and PsbTn (4). The apoproteins of peripheral antennae surrounding the PSII core are encoded mainly by six different Lhcb genes (5). These Lhcb proteins associate with pigments to form homo- and heterotrimeric major-component LHCII (Lhcb1, Lhcb2, and Lhcb3) (6, 7) and three different monomeric minor LHCII species named CP29 (Lhcb4), CP26 (Lhcb5), and CP24 (Lhcb6), respectively (5) (Fig. 1A).

Fig. 1 Overall structures of C2S2M2-type PSII-LHCII supercomplexes.

(A) Cartoon diagram showing the overall subunit composition of plant PSII-LHCII supercomplex. Only one monomer is shown for clarity. The lipid bilayer membrane is shown in gray. (B) Stacked-C2S2M2 supercomplex viewed from the stromal side. The two PSII-LHCII monomers are separated by the black-dotted line, and the dimeric core is highlighted by the black-dashed circle. Four of the core subunits (D1, D2, CP43, CP47) and the peripheral LHCs are shown in ribbon mode, and the color codes are consistent with those in (A). (C) The arrangement of 12 small membrane-intrinsic proteins (colored cartoon models) in each monomer of the stacked-C2S2M2 supercomplex viewed from the stromal side. (D) Side view of the stacked-C2S2M2 supercomplex. (E) Stacked-C2S2M2 supercomplex viewed from the luminal side. (F) Unstacked-C2S2M2 supercomplex viewed from the luminal side.

In natural environments, the light intensity and quality are constantly changing. Plants adopt regulatory strategies to maximize the efficiency of light capture in low light, while quenching excess energy in high light to minimize photodamage. The antenna size of PSII-LHCII supercomplexes is dynamically adjusted in response to changes in light intensity (8, 9). The major-component LHCII trimers associate with the PSII core (C) through the minor LHCII species in either strong (S-LHCII) or moderate (M-LHCII) mode to form large PSII-LHCII supercomplexes. The C2S2M2 and C2S2M complexes, containing both S-LHCII (S) and M-LHCII (M), are the dominant species in plants acclimated to limited light. The amount of smaller C2S and C2S2 particles increases in high-light–adapted plants (8, 10). Recently, we have solved the 3.2 Å–resolution structure of a spinach C2S2 supercomplex, revealing the detailed relationships of S-LHCII, CP29, and CP26 with respect to the core complex (4). The C2S2 particle is further assembled with M-LHCII and CP24 to form the C2S2M2 supercomplex that allows plants to optimize the photosynthetic rate in low light (10, 11). Previously, a two-dimensional projection map at 12-Å resolution and a 30-Å 3D model of the C2S2M2 particle were reported (2, 12). Nevertheless, the accurate orientation and location of the outmost antenna complexes (CP24 and M-LHCII), their interactions and assembly with C2S2 supercomplex, and the precise energy transfer pathways from the outmost antennae to the core remain unknown. The structure may also be affected by light-induced proton accumulation in the thylakoid lumen that can lead to more than a two-unit decrease in luminal pH (13, 14). Lumen acidification was suggested to induce Ca2+ uptake into the lumen (15) and Mg2+ accumulation in the stroma (16), leading to structural remodeling of grana membranes and probably affecting the assembly of OEC proteins (17, 18). The question of how the change of pH and Ca2+ concentration influences the overall architectures of PSII-LHCII supercomplexes is largely open. To reveal the detailed structure of C2S2M2 supercomplex, as well as the relation between pH and Ca2+ and the architectural variation of PSII-LHCII, we have purified pea (Pisum sativum) C2S2M2 samples under different pH and Ca2+ concentrations and obtained C2S2M2 supercomplexes in stacked (at pH 5.7 in the presence of Ca2+) and unstacked (pH 7.5 without Ca2+) states. Sample purification and characterization are summarized in fig. S1.

The stacked C2S2M2 are megacomplexes with two C2S2M2 supercomplexes facing each other at their stromal surface. A reconstruction of dimeric stacked C2S2M2 megacomplex was limited to 8-Å resolution because of flexibility in the interaction between the two C2S2M2 supercomplexes. Thus, only one C2S2M2 supercomplex was reconstructed at an overall resolution of 2.7 Å (fig. S2). The unstacked C2S2M2, containing one C2S2M2 supercomplex, was solved at an overall resolution of 3.2 Å. In both structures, the region of M-LHCII and CP24 was relatively flexible, and this region was refined separately to a resolution of 3.6 and 3.5 Å (fig. S3 and table S1). Overall, there are 28 and 27 protein subunits in each monomer of the stacked and unstacked C2S2M2 structures, respectively, and both contain 157 chlorophyll (Chl), 2 pheophytin, 44 carotenoid molecules, and numerous other cofactors and lipids (Fig. 1, B to F, fig. S4, and table S2). We describe the structural details of the stacked C2S2M2 because of its higher resolution and more complete subunit composition.

In the C2S2M2 supercomplex, M-LHCII and CP24 associate with CP29 from one side near CP47, and S-LHCII and CP26 bind to the core from the opposite side near CP43 (Fig. 1B). Among the six Lhcb proteins, CP24 is the smallest (19), and its structure has not been reported. CP24 has a crucial role in the assembly of the C2S2M2 supercomplex as its absence altered the supercomplex macroorganization (10, 11) and the efficiencies of photosynthesis and photoprotection (11, 20). In the cryo–electron microscopy (EM) structure, CP24 adopts an overall fold similar to those of other Lhcs, but has a shorter C terminus and a longer AC loop with two inserted fragments named IL1 and IL2 (Fig. 2 and fig. S5). Eleven chlorophylls are observed in the CP24 structure (fig. S6, A to C). Their identities are tentatively assigned on the basis of the previous biochemical data (19). In addition, three carotenoid molecules are located at the L1, L2, and N1 sites in CP24. Although previous results indicated that reconstituted CP24 only binds two carotenoids at L1 and L2 sites (19, 21), the pigment molecule at the N1 site shows well-defined cryo-EM density characteristic of a carotenoid (Fig. 2C). CP24 and CP29 form a heterodimer, closely resembling the Lhca3-a2 and Lhca4-a1 dimer of photosystem I (fig. S6D) (22, 23). The carotenoid at the N1 site in CP24 is stabilized at the interface within the CP24-CP29 heterodimer and superposes well with the β-carotene molecule bound to the interface of Lhca dimers (fig. S6D). The N1 site in Lhc proteins can bind either neoxanthin (in Lhcbs) (4, 24, 25) or β-carotene (in Lhcas) (23). Because CP24 lacks the Tyr residue (fig. S5) essential for binding the C3′-hydroxyl group of the neoxanthin in other Lhcbs (26), the N1 carotenoid of CP24 is tentatively assigned as a β-carotene. The other two carotenoid molecules at L1 and L2 sites of CP24 are assigned as lutein and violaxanthin, respectively (19) (fig. S6E and table S3).

Fig. 2 Structure of CP24.

(A) Cartoon representation of CP24 with bound pigments. The chlorophylls and carotenoids are shown as sticks and colored differently. Chl a, green; Chl b, blue; lutein, orange; violaxanthin, yellow; β-carotene, pink. (B) Structural superposition of CP24 (yellow) with other Lhcb proteins, LHCII (green), CP29 (orange), and CP26 (purple-blue). The two inserted fragments in the AC loop of CP24 are indicated as IL1 and IL2. (C) Cryo-EM density of the carotenoid molecule at N1 site in CP24.

Lhcb3 and CP24 are the most recent members of the Lhc family, having evolved after the divergence between land plants and algae (27). They are present only in the C2S2M2 supercomplex, and Lhcb3 is located exclusively in M-LHCII (2, 12). The overall structure of M-LHCII is highly similar to that of S-LHCII, but the three monomers of M-LHCII are not equivalent because of their distinct local environments (Fig. 3A and fig. S7). Cryo-EM density reveals features that match the residues of Lhcb3 on monomer-3 (the one in contact with CP24 and CP29 simultaneously) of M-LHCII (fig. S7B). This assignment of Lhcb3 is consistent with the previous prediction that Lhcb3 is the subunit facing CP24 (2, 28). The structural variations of Lhcb3 and Lhcb1/b2 are mainly in the N terminus, AC loop, and BC loop regions (fig. S7C). Unlike Lhcb1 and Lhcb2 that can form homotrimers, Lhcb3 does not form a homotrimer (6, 29), which might result from the different conformation of the N terminus and the AC loop in Lhcb3 compared to Lhcb1/b2 (fig. S7D). In addition, Lhcb3 has a one-residue insertion at the BC loop, allowing it to form close contacts with CP24 at the luminal side (fig. S7E). For plants lacking Lhcb3, there is a rotational shift of the M-LHCII formed by Lhcb1/b2 (28), underscoring the crucial role of Lhcb3-CP24 interactions in determining the specific orientation of M-LHCII in the C2S2M2 supercomplex.

Fig. 3 The locations and interactions of peripheral antenna complexes.

(A) Surface representation of M-LHCII, S-LHCII′, CP29, and CP24, viewed from the stromal side. The three monomers of M-LHCII are labeled as 1, 2, and 3 for monomers 1, 2, and 3, and monomer-3 is assigned as Lhcb3. M-LHCII is located at the outmost region of the supercomplex and is rotated clockwise by about 20° with respect to the S-LHCII′. (B to F) Interactions between CP24 and CP29 (B), between CP24 and Lhcb3 of M-LHCII (C), between monomer-1 of M-LHCII and S-LHCII′ (D), between CP29 and monomer-1 of M-LHCII (E), and between CP29 and Lhcb3 of M-LHCII (F). Subunits are shown in different colors. The two inserted fragments (IL1 and IL2) in the AC loop of CP24 are highlighted in green. The structural elements and pigment molecules involved in the intersubunit interactions are indicated. The hydrogen bond interactions in (C) are shown as red-dashed lines in the region highlighted with the dashed box.

M-LHCII associates with CP24-CP29 heterodimer and S-LHCII′ in the C2S2M2 supercomplex. Whereas CP24 forms extensive close contacts with both CP29 and M-LHCII (Fig. 3, A to C), the interactions between M-LHCII and S-LHCII′ (Fig. 3D) or between M-LHCII and CP29 (Fig. 3, E and F) are less extensive. IL1 from the AC loop of CP24 interacts with the N-terminal region of CP29, and IL2 is hydrogen bonded with the helix A region of Lhcb3 at the stromal side. At the luminal side, the BC loop of CP24 simultaneously interacts with the C-terminal region of CP29 and the BC loop of Lhcb3 (Fig. 3, B and C). In addition, the carotenoid at the N1 site of CP24 is sandwiched by Chls a601 and a611 from CP29 (fig. S6D), and thus reinforces the intermolecular interaction. Our structure clearly shows the pivotal role of CP24 in linking M-LHCII to CP29 (11).

The 2.7-Å resolution structure enables us to construct the entire pigment network and complete the potential energy transfer pathways in plant PSII. As shown in Fig. 4, the excitation energy transferred by CP24 may be received by CP29 first and then relayed to CP47. CP24 has a Chl b–rich region near helix C, which is close to CP29. At the stromal side, the excitation energy could be transferred from b608CP24 and b609CP24 to a601CP29, which have Mg-to-Mg distances of 17.3 and 17.9 Å. At the luminal side, b606CP24-b614CP29 is the closest interfacial chlorophyll pair, with an Mg-to-Mg distance of 16.9 Å (Fig. 4B). The outmost M-LHCII could also transfer absorbed light energy to CP29 mainly through Lhcb3. Chls a611 from both Lhcb3 and CP29 make the nearest interfacial chlorophyll pair at the stromal surface, with an Mg-to-Mg distance of 16.8 Å, and form a major stromal-side pathway for M-LHCII to transfer the excitation energy. At the luminal side, energy transfer may be achieved from Chl a614Lhcb3 to Chl a613CP29 (Mg-to-Mg distance of 21.2 Å) (Fig. 4C). M-LHCII may also guide its excitation energy to S-LHCII′ through its monomer-1. The Chl a612 pair from M-LHCII and S-LHCII′ has an Mg-to-Mg distance of 20.0 Å, and the Chl pair a610monomer-1/M-LHCII-a611S-LHCII′ has a shorter distance of 19.2 Å. They may serve as alternative stromal-side energy transfer pathways. At the luminal side, Chl a604monomer-1/M-LHCII may transfer excitation energy to a614S-LHCII′ with an Mg-to-Mg distance of 20.6 Å. In addition, the Chl b605 pair from monomer-1 of M-LHCII and S-LHCII′ has an Mg-to-Mg distance of 19.0 Å (Fig. 4D), and both form close chlorophyll pairs with a604CP29 at Mg-to-Mg distances of 19.5 (Fig. 4E) and 17.9 Å, respectively. Curiously, despite the tight interactions between M-LHCII and CP24, their pigments are separated at larger distances. The closest chlorophyll pairs between M-LHCII and CP24 are a614Lhcb3-b606CP24 at the luminal side and a612Lhcb3-b608CP24 and a612Lhcb3-a610CP24 at the stromal side, with Mg-to-Mg distances of 23.9, 19.5, and 23.5 Å, respectively. On the basis of our structures, the excitation energy transfer from the outmost antennae to the core likely occurs mainly through CP29 and S-LHCII′, and subsequently the energy can be further transferred to the inner antennae CP47 and CP43 (4).

Fig. 4 The pigment arrangement and potential energy transfer pathways within the C2S2M2 supercomplex.

(A) Distribution pattern of chlorophylls within the C2S2M2 supercomplex at the stromal and luminal sides. For clarity, the chlorophylls at the luminal side are omitted on the left side of the central black-dashed line, while those at the stromal side are omitted on the right half. Different peripheral Lhc proteins are separated by black-dotted lines and labeled. The monomers 1, 2, and 3 of M-LHCII and the monomers A, B, and C of S-LHCII are labeled as 1, 2, and b3 and A, B, and C. The potential energy transfer pathways from the outmost M-LHCII and CP24 to the adjacent Lhc complexes are indicated by red arrows, while the other pathways are indicated by brown arrows. (B to E) The potential energy transfer pathways from CP24 to CP29 (B), from Lhcb3 of M-LHCII to CP29 (C), from monomer-1 of M-LHCII to S-LHCII′ (D), and from monomer-1 of M-LHCII to CP29 (E). Chls a and b are colored green and blue, respectively. The interfacial chlorophylls involved in the intermolecular energy transfer are highlighted as sticks and labeled. The Mg-to-Mg distances (Å) between two adjacent interfacial chlorophylls are indicated in red.

Superposition of the stacked and unstacked C2S2M2 supercomplexes reveals differences between them, potentially arising from the different purification conditions. The most pronounced differences are found at the luminal extrinsic proteins. The stacked C2S2M2 binds PsbO, PsbP, and PsbQ, but not PsbTn. Meanwhile, the unstacked C2S2M2 binds PsbO and PsbTn, with PsbP and PsbQ missing (Fig. 1, E and F). Removal of PsbP and PsbO under alkaline pH inhibits the water-splitting reaction (30). Our results indicate that PsbP and PsbQ detach from PSII at mild alkaline pH, but PsbTn behaves in an opposite manner. PsbO consists of two domains with a β-barrel core and an extended head, as defined previously (31). The head domain of PsbO is well structured in the stacked C2S2M2 but becomes disordered in the unstacked structure (fig. S8, A to C). The head domain interacts with the C-terminal region of D1, which coordinates Mn4CaO5 and a chloride ion in the stacked C2S2M2. PsbP interacts with the head domain of PsbO and stabilizes the C-terminal tails of D1 and D2 through a loop region (fig. S8D). The absence of PsbP in the unstacked C2S2M2 may lead to reduced stability of the PsbO head domain and the region around Mn4CaO5 cluster. Consistent with this, the unstacked C2S2M2 has relatively disperse density for the Mn4CaO5 cluster as well as its coordinating residues (fig. S8E) and shows lower oxygen-evolving activity compared to the stacked-C2S2M2 sample (fig. S1B). The N-terminal region of PsbP (PsbP-N) is traced in the stacked-C2S2M2 structure. It extends ~20 Å to form multiple interactions with PsbE and PsbF of cytochrome b559 at the luminal side (fig. S8F). In the unstacked-C2S2M2 structure, PsbTn is involved in the interaction with PsbE (fig. S8G).

A slight pivot of the M-LHCII and CP24 with respect to the C2S2 region is apparent in the stacked-C2S2M2 structure compared to the unstacked one (fig. S9), indicating the high mobility of the peripheral antenna complexes in the C2S2M2 particle. This mobility might facilitate detachment of the peripheral M-LHCII and CP24 to down-regulate the efficiency of light-harvesting under high-light conditions. The structures do not include PsbS, a PSII subunit that is essential for photoprotection of plants (32) and was suggested to induce the dissociation of the peripheral subcomplex M-LHCII–CP24–CP29 (33). A wide cleft between the CP24-CP29 heterodimer and the core in the C2S2M2 structures may offer a potential binding site for PsbS (fig. S10). In summary, the high-resolution structures of the C2S2M2-type PSII-LHCII supercomplex at two different states serve as the basis for understanding the subunit assembly and energy transfer within the supercomplex and shed new light on the role of the outer antennae complexes in regulating the light-harvesting capacity and the function of the extrinsic proteins in modulating the oxygen-evolving activity of plant PSII.

Supplementary Materials

Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References (3445)

References and Notes

  1. Acknowledgments: We thank J. Zhang and X. Zhao for their assistance in preparing thylakoid samples. Cryo-EM data collection was carried out at the Center for Biological Imaging, Core Facilities for Protein Science, at the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS). We thank X. Huang, G. Ji, Z. Guo, B. Zhu, D. Fan, F. Sun, and other staff members at the Center for Biological Imaging (IBP, CAS) for their support in data collection; L. Niu and X. Ding for mass spectrometry; J. Li for assistance in fluorescence measurement; L. Kong for cryo-EM data storage and backup; and K. Wang, Y. Yin, and C. Yang (Institute of Botany, CAS) for high-performance liquid chromatography analysis. The project was funded by the Strategic Priority Research Program of CAS (XDB08020302, XDB08030204), the Key Research Program of Frontier Sciences of CAS (QYZDB-SSW-SMC005), the National Key R&D Program of China (2017YFA0503702, 2017YFA0504700), National 973 project grant 2011CBA00900, and National Natural Science Foundation of China (31570724, 31270793). Z.L. and X.Z. received scholarships from the National Thousand (Young) Talents Program from the Office of Global Experts Recruitment in China. The atomic coordinates of the PSII-LHCII supercomplexes have been deposited in the Protein Data Bank with accession codes 5XNL (for stacked C2S2M2), 5XNM (for unstacked C2S2M2), 5XNN (for M-LHCII–CP24 from the stacked C2S2M2), and 5XNO (for M-LHCII–CP24 from the unstacked C2S2M2). The cryo-EM maps of these supercomplexes have been deposited in the Electron Microscopy Data Bank with accession codes EMD-6741, EMD-6742, EMD-6743, and EMD-6744 for the stacked C2S2M2, unstacked C2S2M2, and the M-LHCII–CP24 from the stacked and unstacked C2S2M2. W.C., Z.L., X.Z., and M.L. conceived the project; X.S., P.C., and X.W. prepared the samples; X.S. and X.W. collected the data; X.S., P.C., and M.L. performed the sample characterization; J.M., D.Z., and X.Z. processed the cryo-EM data and reconstructed the cryo-EM maps; P.C. and X.W. built and refined the structure models; P.C., Z.L., X.Z., and M.L. analyzed the structures; Z.L., X.Z., and M.L. wrote the manuscript; all authors discussed and commented on the results and the manuscript.
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