RESEARCH ARTICLE SUMMARY◥

PHOTOSYNTHESIS

Structural basis for blue-green lightharvesting and energy dissipationin diatomsWenda Wang*, Long-Jiang Yu*, Caizhe Xu, Takashi Tomizaki, Songhao Zhao,Yasufumi Umena, Xiaobo Chen, Xiaochun Qin, Yueyong Xin, Michihiro Suga,Guangye Han, Tingyun Kuang†, Jian-Ren Shen†

INTRODUCTION: Photosynthetic organismscontain light-harvesting antenna systems togather light energy required for driving photo-chemical reactions. Diatoms are a group ofeukaryotic algae found in fresh water andoceans throughout the world that help formthe basis of ocean primary productivity by fix-ing massive amounts of carbon dioxide intoorganic carbon. Diatoms are well adapted tothis environment in that they contain light-harvesting antennas with exceptional light har-vesting and photoprotection capabilities, calledfucoxanthin (Fx) and chlorophyll (Chl) a/c-binding proteins (FCPs). FCPs contain the pig-ments Chl c and Fx, which enable them toabsorb light in the blue-green region that is

available under water but not effectively usedby organisms that contain exclusively Chl a/b.These pigments also confer on FCPs a robustenergy-quenching system necessary to thrivein the surface layer of the ocean, an environ-ment with constantly changing light.

RATIONALE: FCP proteins belong to the sup-erfamily of transmembrane light-harvestingcomplex (LHC) proteins with low sequencesimilarity to the main Lhca (LHCI) and Lhcb(LHCII) subunits of the green lineage organ-isms. The structures of LHCI and LHCII fromhigher plants, and the structure of LHCI froma red alga, previously revealed the binding sitesfor pigments in these antenna proteins. This

information was not yet known for FCPs,which limited understanding of the mechanismof light absorption in the blue-green regionand energy transfer and dissipation.

RESULTS: We solved the x-ray crystal struc-ture of a dimeric FCP from a pennate diatomPhaeodactylum tricornutum at 1.8-Å resolu-tion. The FCP was purified as a dimer, and thestructure showed that twomonomers are heldtogether by interactions between their trans-membrane C helices. This differs from the pre-dominant organization of trimers found in the

major LHCII of the green-lineage organisms. EachFCPmonomer binds nineChls and seven Fxs; thenumber of Chls is muchless than the typical 14Chls,whereas that of Fxs is

greater than the three to four carotenoids foundin LHCI and LHCII, resulting in amuchhigherFx/Chl ratio in FCP than those in LHCI andLHCII. Among the Chls, two are Chl c locatedat two sides of the transmembrane helices Aand B, and they are in close interaction withtwo nearby Chls a and one Fx, respectively.This indicates fast energy coupling of Chl cnot only with Chl a but also with Fx. Each Fxis surrounded by one or more Chls, suggest-ing efficient energy transfer between themand also efficient dissipation of excess energyunder high light conditions through the abun-dant Fxs. The binding environment of thetwo end groups of each Fx showed differenthydrophilicities within the protein scaffold,suggesting differences in their preferred ab-sorption region of the blue-green light. Onediadinoxanthin (Ddx) molecule is assignedto a position close to the monomer-monomerinterface because of its weak electron density,suggesting its easy dissociation from the apo-protein and possible involvement in the Ddx-deepoxidation cycle that functions in energydissipation.

CONCLUSION: The FCP structure revealed anetwork of Chls a/c and Fxs that enables ef-ficient blue-green light harvesting and energydissipation in diatoms. The ligand structureand binding environment of each pigment re-vealed in this study will enable detailed studieson the absorption properties of the individualpigments, energy transfer pathways anddynam-ics, and excess energy dissipation mechanismsin this group of antennas, by both theoreticalcalculations and time-resolved spectroscopicapproaches.▪

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Wang et al., Science 363, 598 (2019) 8 February 2019 1 of 1

The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: kuangty@ibcas.ac.cn (T.K.);shen@cc.okayama-u.ac.jp (J.-R.S.)Cite this article as W. Wang et al., Science 363, eaav0365(2019). DOI: 10.1126/science.aav0365

Structure of a FCP.The FCP proteins are found in thylakoid membranes of diatoms and relatedalgae and function as light-harvesting antennas in these organisms.They bind, in addition toChl a commonly present in oxygenic photosynthetic organisms, pigments Chl c and Fx, enablingthem to harvest blue-green light, which can penetrate into water more efficiently. The structurereveals close interactions between Chls and Fxs, suggesting efficient energy transfer anddissipation among these pigments.

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RESEARCH ARTICLE◥

PHOTOSYNTHESIS

Structural basis for blue-green lightharvesting and energy dissipationin diatomsWenda Wang1,2*, Long-Jiang Yu2*, Caizhe Xu1,3, Takashi Tomizaki4, Songhao Zhao1,3,Yasufumi Umena2, Xiaobo Chen2, Xiaochun Qin1, Yueyong Xin2, Michihiro Suga2,Guangye Han1, Tingyun Kuang1†, Jian-Ren Shen1,2†

Diatoms are abundant photosynthetic organisms in aquatic environments and contribute40% of its primary productivity. An important factor that contributes to the successof diatoms is their fucoxanthin chlorophyll a/c-binding proteins (FCPs), which haveexceptional light-harvesting and photoprotection capabilities. Here, we report the crystalstructure of an FCP from the marine diatom Phaeodactylum tricornutum, which revealsthe binding of seven chlorophylls (Chls) a, two Chls c, seven fucoxanthins (Fxs), and probablyone diadinoxanthin within the protein scaffold. Efficient energy transfer pathways canbe found between Chl a and c, and each Fx is surrounded by Chls, enabling the energytransfer and quenching via Fx highly efficient. The structure provides a basis for elucidatingthe mechanisms of blue-green light harvesting, energy transfer, and dissipation in diatoms.

Photosynthetic organisms contain light-harvesting proteins in which pigments arepositioned to absorb and funnel light tothe photosystem core complexes (1–4) toinitiate a series of charge separation and

electron transfer reactions. Excess light absorbedby the light-harvesting pigments is known tocause photodamage (1, 5–7), which may becomesevere in environments with constantly chang-ing light intensity (8). Several photoprotectionstrategies have evolved to dissipate excess energyin photosynthetic organisms (1, 3, 8–11). Diatomsare a group of photosynthetic algae distributedglobally in the oceans and fresh water and con-tribute ~20% of the global primary production(12–14). The light-harvesting antenna of diatomsare fucoxanthin-chlorophyll a/c proteins (FCPs),which have exceptional light harvesting andphotoprotection capabilities (3, 4, 15) and maycontribute to diatoms’ success in environmentswith exposure to intense and variable light.Fucoxanthin (Fx) and chlorophyll (Chl) c pro-

vide an orange-brown color to diatom FCPs, al-lowing them to absorb light in the blue-greenregion (1, 2, 4, 16), which penetrates to deeper

water but is not effectively used by the green-lineage photosynthetic organisms (1, 2, 4). Be-cause of the constant circulation of water in thesurface layer of ocean, diatoms can experienceshifts between weak and strong light in a shortperiod of time (11, 17, 18). Diatoms deal withstrong light through a nonphotochemical quench-ing (NPQ) system, which enables under intenselight dissipation of the excess absorbed energyinto heat. DiatomFCPs display robust NPQ, whichoperates under intense illumination (11, 17–20)and provides protection from photodamage(21–23). The diatom NPQ system is known toinvolve the diadinoxanthin-diatoxanthin (Ddx-Dtx) xanthophyll cycle (3, 19, 20, 24) manifestedby conversion of Ddx to Dtx by the Ddx de-epoxidase under high light and its reverse reac-tion by the Dtx epoxidase under weak light ordark (1, 3, 20). It is not clear, however, to whatextent other factors also contribute to the NPQin diatoms.Diatom plastids originated from endosymbio-

sis of an ancestral red alga (13), and their FCPsbelong to the superfamily of light-harvesting com-plex (LHC) proteins with low sequence similar-ities to the main Lhca (LHCI) and Lhcb (LHCII)proteins of the green lineage organisms (2, 25).The major subunits of diatom FCPs are encodedby lhcf genes, which have more than 10 genes ineach diatom species (26, 27). The energy harvest-ing and dissipation features of FCPs are remark-ably different from those of LHCs found in redalgae and green plants (5, 12–16) because of thebinding of different Chls and carotenoids (Chl cand Fxs) and likely also due to different arrange-ments of the pigments. The sequences of lhcfgenes are highly similar, and the FCP proteins

produced from these genes are also consideredto have similar structure and properties, includ-ing binding of similar numbers of pigments(2, 22, 28). Biochemical and biophysical studieshave been conducted on the light harvesting, en-ergy transfer, and dissipation functions of FCPs(3, 11, 15–25, 29). However, the arrangement ofthe pigments involved in these reactions is notcurrently known, which limits our understand-ing of these processes at a molecular level.

ResultsOverall structure of the FCP

Here, we report the x-ray crystal structure of anFCP from a widely distributed, marine pennatediatom Phaeodactylum tricornutum at 1.8-Å reso-lution (Fig. 1, figs. S1 and S2, and table S1). ThisFCP was isolated in a homodimeric form (Fig.1 and fig. S3) and corresponds to the product oflhcf3 or lhcf4 genes registered in the NationalCenter for Biotechnology Information (NCBI)database (these two genes code for identical se-quences), according to the results of N-terminalsequencing and mass spectrometric analyses.Phylogenetic analysis showed that the Lhcf4protein is similar to both LHCI and LHCII pro-teins, with several conserved residues for thebinding of Chls (fig. S4A), and higher similaritieswere found among the different Lhcf proteins,especially among Lhcf1 to Lhcf12 (fig. S4B). Weconfirmed that the dimeric form of the isolatedFCP is present in solution (fig. S3, A, B, and C).The protein-pigment complex has an apparentmolecular weight of 64 kDa estimated from sizeexclusion chromatography, which is consistentwith sumweight of the apoproteins (~37 kDa perdimer) and pigments (Chls and Fxs, ~27 kDa) fora dimer. The isolated complex is much smallerthan the LHCII trimer (~145 kDa) isolated fromthe green algaBryopsis corticulansunder similarconditions (fig. S3C).Within the crystal structure, two FCP mono-

mers meet at a dimer interface between the C-helices (Fig. 1). Two Chl a406 molecules inserta part of their tetrapyrrole rings into the gapspace of the two helices C to form hydropho-bic interactions, and the phytol tails of the twoChl amolecules also interact with two Ddxmol-ecules to keep them at the position close to themonomer-monomer interface (Fig. 1D). In addi-tion, Arg104 of eachmonomer is hydrogen-bondedwith Ser100 of the adjacentmonomer (Fig. 1D andfig. S5, E and F) at the stromal surface. Theseinteractions thus held the two monomers toform a dimer.Each FCPmonomer contains seven Chls a, two

Chls c, seven Fxs, one Ddx, two calcium cations,one phosphatidyl-glycerol, and one digalactosyl-diacylglycerol. In addition, four n-octal-b-D-thioglucoside (OTG) and one n-dodecyl-a-D-maltopyranoside (a-DDM) used for solubilizationand crystallization were found at the lumenalside and close to Chls and carotenoids, suggest-ing their possible stabilization roles for the FCPduring purification and crystallization.The secondary structure of each monomer

is similar to those of Lhca (or Lhcr) and Lhcb

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1Photosynthesis Research Center, Key Laboratory ofPhotobiology, Institute of Botany, Chinese Academy ofSciences, 100093 Beijing, China. 2Research Institute forInterdisciplinary Science, Graduate School of Natural Scienceand Technology, Okayama University, 700-8530 Okayama,Japan. 3University of Chinese Academy of Science, YuquanRoad, Shijingshan District, Beijing 100049, China. 4PhotonScience Division, Laboratory for Macromolecules andBioimaging (LSB), Paul Scherrer Institut, 5232 Villigen-PSI,Switzerland.*These authors contributed equally to this work.†Corresponding author. Email: kuangty@ibcas.ac.cn (T.K.);shen@cc.okayama-u.ac.jp (J.-R.S.)

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proteins (Fig. 2A and figs. S4A and S5) (30–35);however, the N- and C-termini and loop regionsare shorter in the FCP. This is reflected by thelarge differences in the root mean square devia-tions between the Ca atoms of Lhcf4 (FCP) andLhcb1, which range from 0.7 to 1.2 Å for thethree transmembrane helices but from 9.8 to11.0 Å for the N- and C-termini and loop regions.In the N terminus, a large loop region extendedfrom the beginning of helix A in both Lhca andLhcb at the stromal surface was changed to amuch shorter loop in Lhcf4, whereas in the Cterminus, helix D found at the lumenal surfacewas absent in Lhcf4 (Fig. 2A and figs. S4A andS5). As a result, the characteristic N-terminal“WYGPDR” motif and a C-terminal Trp residuecrucial for the LHCII trimer formation (31–33)are absent in this Lhcf4 (fig. S4A), which mayaccount for why Lhcf4 forms a homodimer andno trimer is found in the present study. In ad-

dition, helix C of Lhcf4 is more tilted against themembrane plane in part to facilitate the forma-tion of the dimer through hydrophobic inter-actions between them, making it shifted relativeto its counterpart in Lhca and Lhcb (fig. S5D).Hydrogen bonds and salt bridges contribute tothe structures of the loop regions in both strom-al and lumenal sides and around the calciumion in the lumenal side (fig. S6). Acidic residueslocated at the lumenal surface (fig. S6) may beinvolved in pH-induced conformational changesduring energy dissipation (8–10, 36, 37).

Arrangement of Chls and energy transfer

FCP contains two Chls c in addition to sevenChls a (Figs. 1, A to C; 2B; and 3 and table S2);the Chl a/c ratio of 3.5 is close to the ratio of 4 asdetermined with biochemical analyses for thesamples used in this study (fig. S7). The totalnumber of Chls in an FCPmonomer, nine, is far

lower than the typical 14 found in most LHCIIand LHCI monomers (table S2). Four Chls a(a402, a404, a406, and a407) and the two Chls care found in similar positions within the struc-ture of LHCII (and LHCI) (Fig. 2B and fig. S8).The other three Chls a have different positions ororientations with those in LHCII, among which,Chls a405 and Chl a409 are found at the lumenalside close to helices C and A, and Chl a401 ispresent in the N-terminal loop region (Figs. 2Band4C). Eight Chls in FCP are coordinated by twoHis, three Glu, and three Gln residues, whereasChl a401 is coordinated by a water molecule (fig.S6D and table S3). This coordination pattern islargely different from those of the inner anten-nae of PSII and PSI core (34, 35, 38, 39) in whichmost of the Chls are coordinated by His residues.The larger variation of Chl ligands in FCP (andLHCI and LHCII) than that in PSI and PSII coreantennae may reflect the evolution of peripheral

Wang et al., Science 363, eaav0365 (2019) 8 February 2019 2 of 8

Fig. 1. Overall structure of FCP from P. tricornutum at a resolutionof 1.8 Å. (A) Overall structure of the FCP dimer with a view from adirection parallel to the membrane plane. The dashed line in thecenter shows symmetric axis of the two monomers. Protein structurein the left-side monomer is depicted in cyan, whereas that in theright-side monomer is depicted in gray. Letters in red shows thetransmembrane helices. Color codes for cofactors are Chl a, green;Chl c, magenta; Fx, orange; Ddx, marine; phosphatidyl-glycerol, violet;

digalactosyl-diacylglycerol, limon; OTG, pale cyan; a-DDM, light blue.(B) Arrangement of the pigments (Chls and Fxs) in the FCP dimerwith the same view as in (A). (C) The structure of an FCP monomer,including water molecules (light pink) and two calcium ions (light blue)located in the stromal side. (D) Monomer-monomer interactionsbetween the two C helices with a view from the stromal side. Hydrogenbonds are depicted in dashed lines in magenta, whereas hydrophobicinteractions are depicted in yellow dashed lines.

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antenna proteins to cope with differences in en-vironment. The absence of Chl b in FCP maytherefore be a result of independent diversifica-tion of FCP from LHCII in the green lineage toaccommodate binding sites for the differentpigments such as Chl c and Fx, which extendedits capacity to absorb different qualities of light(3, 11, 15).The two Chls c are located in the two sides

of the two crossing transmembrane helices Aand B and are coordinated by His39 (Chl c403)and Gln143 (Chl c408), respectively (Fig. 3). Thetwo polar C-17 propionic acids of these Chls cinteract with alkaline residues Arg31 and Lys136,respectively, through ionic bonds, whereas theirC-173 carboxyl oxygen is strongly hydrogen-bonded to the hydroxyl group of the cyclohexane(C) end groups of Fx306 and Fx307 (Fig. 3, B andC, and table S3). The presence of these hydro-

philic groups surrounding the tetrapyrrole ringsof the two Chls c is in agreement with a higherhydrophilicity of Chl c than Chl a owing to thelack of the long phytol tail (40). The close rela-tionship of Fx306 and Fx307 with the two Chls cimposes restraints on the species of Chls that canoccupy these positions because the phytol tails ofChl a will apparently be in conflict with the twoFxs. Because carotenoids corresponding to Fx306and Fx307 are absent in LHCI and LHCII (Fig.2C), this may account for why these positionsbind Chl a in the green lineage but Chl c in FCPs.In addition to the hydrophilic groups, each of thetwoChl cmolecules are in close interactionswithtwo Chl a molecules (Fig. 3, B and C).The Chls in FCP are distributed in layers on

the lumenal and stromal sides of the thylakoidmembrane (Fig. 4). In the stromal layer, six Chls(four Chls a and two Chls c) form two coupled

Chl a-c-a clusters (a402-c403-a406, and a401-c408-a407) in each monomer (Fig. 4, A and C),amongwhich, Chls c403 and c408 in each clusterare strongly coupled with Chls a406 and a401,with partial overlaps of their tetrapyrrole ringsand a closest p-p distance of 3.4 and 3.9 Å, re-spectively (Fig. 3, B and C, and table S4). On theother hand, the p-p distances between the tetra-pyrrole rings of Chls c403-a402 and Chls c408-a407 are at 5.6 and 6.1 Å, respectively (table S4).These close p-p distances enable fast and efficientexcitation energy transfer between Chl c andneighboring Chl a at two directions within thetwo Chl a-c-a clusters, explaining the speed at60 to 100 fs and the efficiency at 100% of en-ergy transfer between Chl a and Chl c previ-ously determined with time-resolved spectroscopy(15, 40, 41). The p-p distance between the twoclusters is 9.6 Å within the same FCP monomer

Wang et al., Science 363, eaav0365 (2019) 8 February 2019 3 of 8

Fig. 2. Comparison of the structures between FCP and LHCII.(A) Superposition of the structures of an FCP monomer (Lhcf4, red)with LHCII [Lhcb1, Protein Data Bank (PDB) code 1RWT, marine] (31).The apoproteins are depicted in ribbon, and all of the cofactors areremoved for clarity. The areas encircled by dashed lines indicatestructures with large differences. The helices are labeled with red

letters. (B) Superposition of the Chls of FCP with those of LHCII.Chls a and Chls c of FCP are depicted in green and magenta, andChls a, b of LHCII are depicted in blue and light gray, respectively.(C) Superposition of the carotenoids of FCP with those of LHCII. Fxsare depicted in orange, Ddx in marine, lutein in yellow, vioxanthin inlime, and neoxanthin in purple.

Fig. 3. Binding environments of two Chl cmolecules. (A) Overall positionand coordinating environments of the two Chl c molecules. Coordinating bondsfor the two Mg2+ ions are depicted in solid lines, whereas hydrogen bonds are

depicted in dashed lines. Color codes used are the same as in Fig. 1. (B and C)InteractionsofChl c408 (B) andChl c403 (C)with nearbyChl a andFxmolecules.Distances for hydrogen bonds and p-p interactions are given in angstroms.

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(Chl a401-Chl a402), but 6.4 Å with the adjacentmonomer (Chl a406-a406*) (table S4), suggest-ing fast and efficient energy transfer between thetwo monomers in the stromal layer and an ad-vantage for the formation of the dimer. Thesestructural features, in combination with theintrinsic characteristics of the pigments, makeChl c an efficient harvester of blue-green andeven yellow light, which is the “green gap”whereChls a and b absorbweakly. The energy absorbedby Chls c is then transferred efficiently to thecoupled Chls a (1, 4).The remaining three Chls (a404, a405, and

a409) are located close to each end of the threetransmembrane helices at the lumenal side (Figs.2B and 4, B and D), with an edge-to-edge dis-tance of ~8.4 Å between Chls a404 and a405 anda much longer distance between Chls a404 anda409 (table S4). The p-p distance between thestromal and lumenal layers is 8.9 Å (Chl a404-a406) and 11.8 Å (Chl a409-a402) in a monomer(Fig. 4B), suggesting a slower excitation energytransfer between the two layers, which is similarto what has been reported for LHCII and LHCI(31–35). Because the seven Chl a molecules inFCP are clearly separated, strong Chl a-c cou-pling will be dominant, whereas Chl a-a couplingwill be rather weak based on the present struc-ture. This organization of the Chl a moleculessuggests similarities in their spectral proper-ties, which is in agreement with previous spec-troscopic results showing that all of the Chls a inFCP have similar energetic levels (15, 28). There-

fore, it would be difficult to determine the trapand exit sites of the absorbed excitation energyon the basis of the excitonic level of individualChls. However, on the basis of the current struc-ture, we propose that the lateral Chl a401 stronglycoupled with Chl c may be responsible for col-lecting absorbed excitation energy and transmit-ting it to the reaction center or adjacent antennas.

Arrangement of FXs and blue-green lightabsorption, energy dissipation

FCP binds seven Fxs and one Ddx (Figs. 2C and5A). By contrast, each of the LHCI and LHCIIsubunits bind only three or four carotenoids (Fig.2C and fig. S8) (30–35). Fx303 and Fx305 bind tothe conserved lutein sites in LHCII, with theirpolyene chains embedded in the grooves formedby the crossing-helices A and B, and their endgroups are more twisted, whereas the remainingfive Fxs and the only Ddx bind in positions inwhich no corresponding carotenoids are foundin LHCII (Fig. 2C and fig. S8). The polyene back-bones of six Fxs are inclined inside the hydro-phobic membrane, and their epoxycyclohexane(E) and cyclohexane (C) end rings are orientedtoward themembrane surfaces (Fig. 5A and tableS5). The remaining Fx304 is located at the stromalsurface, with an angle almost horizontal to themembrane under the “tongue” region of the C-Aloop, whose polyene chain is shielded by sidechains of Ile121 and Phe123 at the stromal side.This carotenoid binding mode has not been ob-served in LHC proteins (30–35, 42), and we sug-

gest that it may be involved in tuning pigmentwavelength.Fx has large solvent effects similar to that

seen in peridinin and siphonaxanthin (43–46):An increase in the polarity of the solvent causes alarge redshift in their absorption due to the pres-ence of the conjugated carbonyl groups in thesecarotenoids. The varied binding environments ofFxs in FCP imply different spectroscopic proper-ties. Because Fx303 and Fx305 are located in thecentral region of themembrane, theymay absorblight predominately in the “blue” region becauseof their hydrophobic binding environment. Fx301and Fx302 are suggested as “green”molecules forthe presence of polar pockets surrounding their C-end groups, whereas Fx306 and Fx307 are pro-posed as “red” molecules for their polar bindingenvironments of both end groups to make theirabsorption largely red-shifted to 500 to 550 nmregion (29, 45–48). In fact, although the E-rings ofFx306 and Fx307 are located at the lumenal sur-face, their C-rings are located inside the mem-brane.However, these two Fxs are close to the twoChl c molecules, with their polyene chains run-ning parallel to the tetrapyrrole rings of the twoChls c at ~3.5 Å and their C-rings hydrogen-bonded to the two propenoic acids of the twoChlsc molecules (table S5) as shown above (Fig. 3, Band C, and table S5), making the environment ofthe C-rings of these two Fxs also rather hydro-philic. Similarly, the distinct Fx304may also be a“red Fx” because its two end groups are locatedat the stromal surface close to the bulk solution

Wang et al., Science 363, eaav0365 (2019) 8 February 2019 4 of 8

Fig. 4. Distribution of Chls in FCP and possible energy transferpathways among them. (A) Distribution of the Chls with a view alongthe membrane plane. The adjacent Chls in the same layers are connectedwith dashed lines, and their center-to-center distances are labeled

(angstroms). (B) The same view as in (A), with the distances betweenthe stromal and lumenal side layers indicated. (C and D) Distribution ofChls with views from the stromal side (C) and lumenal side (D). Colorcodes for the pigments are the same as those in Fig. 1.

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and also to the Chl c molecules. These struc-tural features allow Fx to play the major light-harvesting role, particularly under water, wherelight in the red region useful by Chl a diminishesquickly and more “green” photons arrive (3, 7).The remarkably high Fx/Chl ratio in FCP ef-

fectively results in every Fx being surrounded byone or more Chls (Figs. 3, B and C, and 5, B to G),where p-p interactions between Fxs and the tet-rapyrrole head groups of Chls are found at dis-tances of ~4.0 Å in most cases (table S4). Theseclose contacts enable fast and efficient excitationenergy transfer between Fxs and Chls, which hasbeen shown to be as fast as 75 fs and with anefficiency beyond 90% for the transfer of ab-sorbed green light from Fx to Chl a (15, 41, 48),ensuring Fx to efficiently harvest and use photonenergy in the “green-gap” region. However, en-ergy transfer from Fx to Chl c in the major FCPcomplexes has not been observed so far (48). Ourstructure suggested that this kind of energy trans-fer should be very fast and therefore may haveescaped from detection. On the basis of the cur-rent structure, the energy harvested by Fx306and Fx307 must be transferred to Chl c403/Chl

c408 located between Fx306/Fx307 and Chl a406/Chl a401 (Fig. 3, B and C) before being transfer-red to the Chl a molecules. Thus, time-resolvedspectroscopic studies with a higher time resolu-tion are required to clarify the possible energytransfer from Fx to Chl c.The close association of every Fx with respec-

tive Chls also suggests that the excess energyabsorbed by Chls may be dissipated quickly andefficiently through their nearby Fxs, thus pro-tecting the photosynthetic apparatus from photo-damage (3). Excessive irradiance will producetriplet Chls and reactive oxygen species at thephotosynthetic reaction centers (37), causing pho-todamage to the photosynthetic apparatus. Photo-protection is a mechanism by which the excessenergy is dissipated as heat, protecting photo-systems from damage (3, 37). Because diatomslive under rapidly fluctuating light environmentowing to the rapidmixing in the surface of ocean(3, 7), they have gained a large amount of light-harvesting antenna proteins (FCPs) to harvestenergy effectively under low light conditions.However, when diatoms are brought to the sur-face layer of ocean in a short time (3), the large

amount of FCPswill absorb toomuch light, whichmay cause photodamage. Under such conditions,photoprotection is triggered by the Ddx-Dtx de-epoxidation reaction, transmembrane proton grad-ient, and/or reorganization and conformationalchanges of the FCP complexes (8, 37). In thepresent structure, we assigned one Ddx in thevicinity of the twinned helices C in the inter-face of the two monomers (Figs. 1 and 5). Theelectron density for the Ddx molecule is weak(fig. S2C), suggesting that it may be disorderedor present at low occupancy because of weakinteractions with protein or other ligands. Thisis in agreement with the biochemical analysisof our purified sample showing that there isless than one Ddx per FCP monomer (fig. S7)and may explain why the amount of Ddx ob-tained from biochemical analyses varies widelyin the literature (fig. S7) (15, 16, 28, 49, 50) be-cause Ddx may be easily lost during purifica-tion. The apparent weak binding of Ddx mayfacilitate exchange through the Ddx-Dtx cycle.Acidification of the lumenal space has been

shown to trigger reorganization and/or con-formational changes of the antenna proteins,

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Fig. 5. Distribution of Fxs and Ddx and the possible quenchingsite in the FCP dimer. (A) Side view of the distributionof Fxs with the protein subunits depicted in a surface model.

(B to G) Interactions of the individual Fxs with nearby Chls,with their closest distances for the p-p interactions givenin angstroms.

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initiating another mechanism of energy dissi-pation (8, 37). Several acidic residues (Glu54,Asp64, Glu72, Glu82, and Glu158) were found to beexposed to the lumenal surface in the FCP struc-ture (fig. S6A); these residuesmay be relatedwiththe protonation-induced conformational changesthat function in photoprotection. Among theseresidues, Glu72 and Glu82 are coordinated totwo calcium cations at the lumenal surface (fig.S6B), suggesting that pH-induced reorganization(8, 51, 52) may be modulated by divalent cations.In addition, conformational changes of Fx301and Fx302 may be triggered by protonation ofacidic residues in the BC- and/or C-terminalloops, leading to the quenching of excitationenergy from the nearby Chls similar to thatseenwith the central lutein inLHCII (8–10, 36, 37).Furthermore, Fx301 is physically coupled withChls a401 and a409 (Fig. 5) and therefore alsoprone to undergo conformational changes uponaggregation of FCP, as seen for the correspond-ing neoxanthin in LHCII (8, 11, 15, 37). Thesestructural features indicate that the arrangementof Fxs and Chls in the FCP dimers is optimized toassure the efficient light-harvesting in the blue-green region as well as energy dissipation undera highly fluctuating light environment.

Conclusions

The structure of FCP reveals an arrangement ofChls a/c and Fxs, ensuring efficient blue-greenlight harvesting and energy transfer and dissipa-tion in diatoms, a dominant phytoplankton. Weanticipate that the structural information pro-vided by this study will greatly promote theoret-ical and time-resolved spectroscopic studies toelucidate the energy migration and dissipationmechanisms in this group of antenna proteins.

Materials and methodsCell culture and FCP purification

Cells of the pennate diatom Phaeodactylumtricornutum (FACHB-863, Freshwater AlgaeCulture Collection at the Institute of Hydrobiol-ogy, Wuhan, China) were grown in a sterileartificial seawater F/2 medium at 22°C undercontinuous light (40 mmol photons m−2 s−1) forone week. The cells were harvested and resus-pended in an ice-cooled medium containing20 mM tricine, 10 mM MgCl2, 20 mM KCl, 5%sucrose, pH 7.8 (TMKS buffer), and then dis-rupted by glass beads. Thylakoid membraneswere collected by centrifugation at 100,000 × gfor 20 min at 4°C, followed by solubilization with1% (w/v) n-dodecyl-a-D-maltopyranoside (a-DDM)and at a concentration of 0.5 mg Chl a/ml for30 min on ice based on the method used forisolation of LHCII from a green alga Bryopsis(44) and Cyclotella (53). After centrifugation toremove unsolubilized membranes, the FCP en-riched supernatant (FCP pool) was loaded ontoa Q-Sepharose HP column (GE Healthcare) pre-equilibrated with the TMKS buffer containing0.03% (a-DDM). The column was washed with0.25 M NaCl to remove most of contaminatingproteins, followed by elution with a linear grad-ient of 0.25 to 0.42 M NaCl. The Lhcf4-rich frac-

tion (labeled Lhcf4) was eluted at 0.35-0.38 MNaCl (fig. S3A), which was collected, concen-trated with an AMICON centriprep-50 (cut-offmolecular weight: 50 kDa) filter and then puri-fied with a second Q-Sepharose HP column withthe same conditions as the first column, to in-crease the purity of the FCP. The eluted FCPwas concentrated again and loaded onto a lin-ear sucrose gradient with 5 to 20% sucrosein the TMK buffer containing 0.03% a-DDM,followed by centrifugation at 303,800 × g for16 hours (fig. S3B). Two bands were obtained,among which, the upper band is an FCP mono-mer and the lower band is an FCP dimer basedon their apparent molecular masses analyzed bygel filtration chromatography (fig. S3C, the elu-tion profile for the FCP monomer was not shownfor clarity). The band containing the FCP dimerwas collected and concentrated by precipitationwith 25% PEG1000, and used for crystallization.The oligomerization states of the “FCP pool” sol-ubilized from the thylakoid membranes by thedetergent and the purified FCP were analyzedby gel filtration with a Superdex 200 PC3.2/30column (2.4 ml, GE Healthcare) (fig. S3C). Forcomparison, dissolved FCP crystals and LHCIItrimers from a green alga Bryopsis were alsoanalyzed by the gel filtration (fig. S3C). Theresults showed that the FCP pool before sepa-ration by the ion-exchange chromatography con-tained mainly dimers and monomers, whereasthe purified FCP and re-dissolved crystals had amolecular mass equivalent to an FCP dimer.

Crystallization

Crystallization trials were performed using theoil batch method at 293 K in which, 3.0 mL ofthe FCP-dimer dissolved in 10 mM MES [2-(N-morpholino)ethanesulfonic acid; pH 6.5] at 2.0mgChls ml−1 (Chls a and c) was mixed with an equalvolume of a precipitate solution containing100 mMMES (pH6.5) or Tris [tris(hydroxymethyl)aminomethane; pH 8.5], 22 to 26% PEG 1000,100mMCaCl2, 3% (w/v) n-octal-b-D-thioglucoside(OTG, Anagrade, Anatrace), and then coveredwith 15 mL paraffin oil (Hampton). Crystals ap-peared within 2 to 3 days and reached a max-imum size of 0.20 mm × 0.10 mm × 0.05 mmwithin 5 days, and exhibited a dark-orange colorand elongated rectangular shape (fig. S1, A andB). We confirmed that the FCP dimer in the crys-tal retained the similar absorption and fluores-cence properties before crystallization (fig. S9).For the x-ray diffraction experiments, the crystalswere transferred into a cryo-protectant solutioncontaining 45% PEG 1000, 50 mMMES (pH 6.5)or Tris (pH 8.5), 50 mM CaCl2, 1.5% (w/v) OTGand incubated for 5min, and then flash-frozen ina nitrogen stream at 100 K.

Data collection, processing, structuraldetermination, and refinement

Screening of the resolutions of crystals and nativedata collection were performed at beamlinesBL17U1 of Shanghai Synchrotron Radiation Fac-ility, China (54) and BL41XU of SPring-8, Japan.Several thousands of crystals were screened,

among which the best crystals diffracted to aresolution of 1.63 Å (fig. S1 and table S1). Dif-fraction images were recorded at an X-ray wave-length of 1.0 Å, with an exposure time of 0.1 s anddetectors of ADSC Q315r CCD for BL17U1 andPilatus 6M for BL41XU. The data set was col-lected by rotating the crystal with a 0.2° oscilla-tion angle over a range of 130°. The FCP crystalsobtained belonged to the space group P212121(grown in the pH 8.5 Tris buffer) or C2221(grown in the pH 6.5 MES buffer), with unit celldimensions a = 47.79 Å, b = 123.31 Å, c = 140.71 Å,and a = 47.75 Å, b = 115.72 Å, c = 141.26 Å, re-spectively (table S1). Both types of the crystalsshowed a severe anisotropy in the diffractionintensities in the axis b, so ellipsoidal trunca-tion was performed with the STARANISO server(http://staraniso.globalphasing.org) (55). The pro-cessing results from this server suggested anoverall resolution limit of 1.64 Å with a CC1/2of 0.3, as well as resolutions of 1.64 Å and 1.79 Å,respectively, based on a criteria of mean I/sigma(I) over 1.5 or 2.00. Due to the high anisotropy ofthe diffraction data, the resolutions of differentdirections were suggested to be 1.64 Å, 2.82 Åand 1.72 Å, respectively, for the a, b and c axeswith a criterion of mean I/sigma(I) over 1.50.Structural refinement performed at the later stagesshowed that gap betweenRfree andRwork becamelarger and the Rfree was also increased when theresolution was set to higher than 1.80 Å. Thus, wedetermined the overall resolution of our struc-ture to be 1.80 Å.Single-wavelength anomalous diffraction (SAD)

data sets were collected from a single crystal atbeamline X06DA of the Swiss Light Source at thePaul Scherrer Institute (Switzerland) at an X-raywavelength of 2.075 Å, using a multi axis gonio-meter, PRIGO and a Pilatus 2M dector (56). Dueto the high anisotropy and low isomorphism ofthe crystals, a very high redundancy of the nativeSADdata had to be collected from a single crystalin order to obtain the phase information. Alldiffraction data was processed, integrated andscaled using the XDS Program Package (57).The native SAD data set was indexed and

scaled to 2.7 Å resolution, which showed a re-dundancy of 123 for the whole resolution rangeand 110 for the highest-resolution shell, respec-tively. Based on the SAD data, phase informa-tion was calculated with Crank2 in CCP4i2 (56, 58),which gave rise to 20 apparent anomalous peaksin the anomalous difference Patterson map for adimer. These peaks were later identified as 4calcium atoms, 8 sulfur atoms from methionine,7 magnesium atoms from Chls and 1 sulfur atomfrom OTG in each asymmetric unit. Iterativesubstructure improvement and phasing wereperformed with REFMAC5 and PEAKMAX, andthe handedness of substructures was deter-mined by Solomon in Crank2 of CCP4i2. Den-sity modification was performed with Parrotwith Fourier recycling, and BUCCANEER wasused for combined iterative model buildingwith density modification and phase refine-ment in Crank2 of CCP4i2. Pigments, lipids andcofactors were incorporated and the amino acid

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residues were manually modified using COOT(59). The phase was then extended to the finalresolution of 1.8 Å with the native dataset usingmolecular replacement by PHASER (60). Resi-dues 1 to 166 out of the total 167 residues weretraced in the model. The quality of the structurewas analyzed using Procheck (61). All figureswere prepared using Pymol (62).

Assignment of the lightharvesting cofactors

Chls a and c were identified based on the exist-ence of a phytol chain for Chl a, and the planarityof C-181, C-18, C-17, and C-171 given by the C-18=C-17 double bound for Chl c. While most ofthe Chl a have clearly identifiable phytol tails,Chl a405 has almost no clear electron densityfor the phytol tail and its head group may bebetter fitted with a Chl c; however, based on thepigment analysis it was assigned as a Chl a. Thetwo Chl c sites were further differentiated intoChl c1 and Chl c2, and structural refinement witha loose restraint in the distance between theethyl and ethylene groups of C-8 provided thetentative assignment of Chl c403 as Chl c2 andChl c408 as Chl c1, respectively. The electrondensity for the Ddx molecule was also ratherweak especially in its head group, and this sitecould be occupied with a lower occupancy (oreven by other carotenoids). The orientation ofthe Ddx molecule is assigned with its epoxygroup directed toward the stromal side in thepresent structure. When we reverse the orien-tation of this molecule and performed the struc-tural refinement again, we found that the Rfree

value of the whole structure increased by 0.2%,and the B-factor of the Ddx molecule increasedfrom 87 to 90. Thus, we consider that the currentorientation of the Ddx molecule is consistentwith our electron density map.

Pigment analysis

Pigments were extracted from the FCP samplewith 90% (v/v) acetone and the Chl concentra-tion was calculated as previously described (63).HPLCwas performed with a C-18 reversed-phasecolumn (ϕ 4.6mm, length 250mm, 5 mmparticlesize, Grace, USA) in a Waters e2695 separationmodule equipped with a Waters 2998 photo-diode array detector. The pigments were elutedat a flow rate of 1 ml/min using a 20 min lineargradient with 0 to 100% solvent B (ethyl acetate)mixed with solvent A (methanol:water = 90:10),whichwas then continued isocratically with 100%solvent B for 2 min. Pigments were detected bytheir absorbance at 445 nm.The authentic standard of Chl a was pur-

chased from Sigma. For commercially unavail-able standards (Chl c, Fx. and Ddx), the pigmentswere extracted from the P. tricornutum thyla-koid membranes. The extinction coefficients forChl c (c2 type) and other pigments used weretaken from refs. 5 and 36. Standard curves forthe quantification of pigments were obtainedby plotting the amount of the pigments loadedonto the HPLC column against the area of eachelution peak, with each data point obtained from

the average of three independentmeasurements.For comparisons, other values reported previous-ly for different kinds of samples were summar-ized in fig. S7 (15, 16, 28, 49, 50).

Peptide analyses andspectrophotometric measurements

Protein composition of the FCP samples wasanalyzed using SDS-PAGE with a gel contain-ing 16% polyacrylamide and 7.5 M urea. Foridentification of the FCP subunit, N-terminalsequencing, mass spectrometric analyses andspectrophotometric measurements were per-formed as described previously (44, 64). TheN-terminal sequence obtained was AFEDEL-GAQPPLGFF, which was used together withseveral internal sequences from digested pep-tides to search homologous sequences in theNCBI databases, which gave rise to two sequen-ces encoded by lhcf3 and lhcf4 of P. tricornutum,respectively. These two genes encoded exactlythe same sequence with a total of 167 aminoacid residues. Thus, the FCP subunit was iden-tified to correspond to the product of lhcf3 orlhcf4. Alignment of homologous sequences ac-quired from NCBI database and phylogeneticanalysis were performed with tools provided bythewebsitewww.phylogeny.fr/simple_phylogeny.cgi (65).Absorption spectra were measured at room

temperature with an ultraviolet-visible spec-trophotometer (UV–Vis 2550, Shimadzu, Japan)at 10 mg Chl ml−1 in the TMK buffer containing0.03% a-DDM. Low-temperature (77 K) fluores-cence emission and excitation spectra were mea-sured using a fluorescence spectrophotometer(F-4700,Hitachi, Japan) at a Chl concentration of2 mg Chl ml−1 for FCP dimer in the TMK buffersupplemented with 30% glycerol and 0.03%a-DDM. The spectral sensitivity of the fluores-cence spectrophotometer was corrected usinga light source with a known radiation profile(Hitachi, Japan).

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ACKNOWLEDGMENTS

We thank M. Sang and D. Chen for their help with the culture ofthe diatom cells, N. Matsugaki for help on the collection of theSAD data, the CCP4 workshop team (SPring-8 Japan, 2017) foradvices on analyzing the native-SAD data and phasing, and H. Linfor discussions. Diffraction data at 1.0-Å wavelength wascollected at beamlines BL17U1 of Shanghai Synchrotron RadiationFacility (SSRF, China) and BL41XU of SPring-8 (Japan); NativeSAD data was collected at beamlines BL1A of Photon Factory(PF, Japan) and X06DA of Swiss Light Source at the PaulScherrer Institute, and we thank the staff members of thesebeamlines for their extensive support. Funding: This work wassupported by the National Key R&D Program of China(2017YFA0503700), a Strategic Priority Research Program ofCAS (XDB17000000), a CAS Key Research program for FrontierScience (QYZDY-SSW-SMC003), a National Basic ResearchProgram of China (2015CB150100 to T.K.), and JSPS KAKENHIno. JP17H0643419 of MEXT, Japan (to J.-R.S.). Author contributions:J.-R.S., W.W., and T.K. conceived the project; C.X., S.Z, and W.W.cultured the cells and purified the FCP complex; W.W. and C.X. grewand optimized the FCP crystals; W.W., C.X. and L.-J.Y. conductedthe diffraction experiments and collected the data at 1.0 Å wavelength;W.W., L.-J.Y., Y.U., and T.T. collected the diffraction data for SADphasing; W.W., and L.-J.Y. determined the phase; L.-J.Y., M.S., andW.W. refined the structures; W.W., L.-J.Y., T.K. and J.-R.S. wrote themanuscript; and all authors discussed and commented on the resultsand the manuscript. Competing interests: The authors declarethat they have no competing interests. Data and materialsavailability: Atomic coordinates have been deposited in the PDBunder the accession no. 6A2W, in which four new ligands wereassigned as A86 (Fx), DD6 (Ddx), KC1 (Chl c1), and KC2 (Chl c2),respectively. All other data are presented in the main text orsupplementary materials.

SUPPLEMENTARY MATERIALS

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7 August 2018; accepted 31 December 201810.1126/science.aav0365

Wang et al., Science 363, eaav0365 (2019) 8 February 2019 8 of 8

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Structural basis for blue-green light harvesting and energy dissipation in diatoms

Yueyong Xin, Michihiro Suga, Guangye Han, Tingyun Kuang and Jian-Ren ShenWenda Wang, Long-Jiang Yu, Caizhe Xu, Takashi Tomizaki, Songhao Zhao, Yasufumi Umena, Xiaobo Chen, Xiaochun Qin,

DOI: 10.1126/science.aav0365 (6427), eaav0365.363Science 

, this issue p. eaav0365Sciencehelp transfer and disperse light energy.light-harvesting complexes of plants but have more binding sites for carotenoids and fewer for chlorophylls, which may light that penetrates to deeper water and is not absorbed well by chlorophylls a or b. FCPs are related to thespecialized photosynthetic pigments in this light-harvesting protein. Fucoxanthin and chlorophyll c absorb the blue-green

binding protein (FCP) from a diatom. The structure reveals the arrangement of the−fucoxanthin chlorophyll a/c determined the structure of a et al.energy and either transfer it to photosystems or disperse it as heat. Wang

which absorb light−−chlorophylls and carotenoids−−too much light, which causes damage. Both tasks require pigments Photosynthetic organisms must balance maximizing productive light absorption and protecting themselves from

All the hues, even the blues

ARTICLE TOOLS http://science.sciencemag.org/content/363/6427/eaav0365

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/02/06/363.6427.eaav0365.DC1

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