Author response: The photosystem I supercomplex from a primordial green alga Ostreococcus tauri harbors three light-harvesting complex trimers

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in low light conditions, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). The architecture of the supercomplex exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, an Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and an Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of OtPSI-LHCI-Lhcp supercomplex is likely due to a phosphorylation-dependent mechanism induced by changes in light intensity. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to the weak coupling of the chlorophyll a603-a609 pair in OtLhca1-4. Editor's evaluation This fundamental work represents an important contribution to our understanding of the diversity of photosynthetic mechanisms across the branches of phototrophic life, with the first high-resolution structure (2.9 Å) of a photosynthetic complex from the green alga, Ostreococcus tauri, an ecologically important green alga utilizes a unique antenna complex, Lhcp. The evidence suggests mechanism found here is distinct from the classical antenna state transitions seen in other organisms studied thus far, expanding our knowledge of how photosynthetic systems react to changes in light conditions and leading to a new understanding of the function and evolution of light-harvesting antennas. https://doi.org/10.7554/eLife.84488.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Phytoplankton are the major primary producers in the aquatic environments, and provide organic matter for marine food webs by converting solar energy into chemical energy through photosynthesis. As a member of natural phytoplankton living in the ocean, Ostreococcus tauri is a unicellular green alga widespread in marine environments and crucial for the aquatic ecosystem (Derelle et al., 2006; Palenik et al., 2007). It is also known as the smallest free-living eukaryote (Courties et al., 1994) and belongs to Prasinophyceae, a class of green algae at the near basal position in the evolution of green lineage and most closely related to the first green alga known as ‘ancestral green flagellate’ (Lewis and McCourt, 2004). A prasinophyte-specific Lhc protein named Lhcp was found in O. tauri (Six et al., 2005; Swingley et al., 2010) and Mantoniella squamata (another member of Prasinophyceae) (Jiao and Fawley, 1994; Schmitt et al., 1994). There are two classes of Lhcp (Lhcp1 and Lhcp2) in addition to the common LHC proteins, namely six Lhca proteins (Lhca1-6) and two minor monomeric Lhcb proteins (Lhcb4 and Lhcb5), serving as the peripheral antennae of O. tauri PSI and PSII (Six et al., 2005; Swingley et al., 2010). The Lhcp proteins form a highly abundant antenna complexes in O. tauri (Swingley et al., 2010), and the carotenoid composition of the OtLhcp complexes is largely different from those of LHCIs or light-harvesting complexes II (LHCIIs) in plants and other green algae (such as a core chlorophyte Chlamydomonas reinhardtii) (Minagawa, 2009). While Lhcp, Lhcb (apoproteins of LHCII), and Lhca (apoproteins of LHCI) all belong to the LHC superfamily, Lhcp proteins form a separate clade with characteristics of an ancestral state of LHC proteins instead of belonging to the clades of Lhcb or Lhca (Six et al., 2005). Moreover, the organization of pigment molecules within the Lhcp complexes exhibits distinct features in comparison with plant LHCII according to a previous spectroscopic study (Goss et al., 2000). Under low light (LL) conditions, the Lhcp complexes of O. tauri can assemble with the PSI-LHCI to form a larger PSI-LHCI-Lhcp supercomplex, whereas the amount of PSI-LHCI-Lhcp supercomplex is greatly reduced under high-light (HL) conditions (Swingley et al., 2010). It remains unclear how the Lhcp complexes assemble with PSI and establish energy transfer pathways with the interfacial PSI subunits under LL conditions. The arrangement of various Chl and carotenoid molecules within the Lhcp complexes is also unknown and awaits to be analyzed through further studies. Results and discussion The PSI-LHCI-Lhcp supercomplex To stabilize the photosynthetic supercomplexes from O. tauri during purification process, the detergent-solubilized thylakoid membrane was treated with amphipol A8-35, which is an amphipathic polymer to substitute for detergents, before the sucrose density gradient (SDG) ultracentrifugation (Figure 1a, Figure 1—figure supplement 1a), according to the previous protocol successfully employed to stabilize the PSII supercomplex from C. reinhardtii (Watanabe et al., 2019). Pigment composition analysis of the A3L fraction demonstrates that the most abundant light-harvesting carotenoid is prasinoxanthin (Prx, which is unique to prasinophytes), among other carotenoids of lesser abundance, including dihydrolutein (Dlt) and micromonal (Figure 1—figure supplement 1c, Table 1). Although the typical Chls (such as Chl a and Chl b) and carotenoids (violaxanthin/Vio, 9’-cis-neoxanthin/Nex and β-carotene) found in green plants were also present in O. tauri, lutein (a major carotenoid found in plant LHCII) was not detected. Polypeptides for the PSI core and its peripheral antennae in the A3 and A3L fractions were characterized by SDS-PAGE (Figure 1—figure supplement 1b) and mass spectrometry analysis (Supplementary file 1a). These findings indicate that A3 is primarily composed of polypeptides for PSI-LHCI supercomplex, whereas A3L includes Lhcp1 and Lhcp2 alongside the constituents present in A3, along with comigrated PSII polypeptides, which is essentially in agreement with a previous report (Swingley et al., 2010). Figure 1 with 4 supplements see all Download asset Open asset Characterization of A3 and A3L fractions. (a) Sucrose density gradient showing four major bands corresponding to free LHCs (A1), PSI-LHCI supercomplex (A3), PSI-LHCI-Lhcp supercomplex and PSII-Lhcp supercomplex (A3L), and other complexes (A4). (b) 77 K steady-state fluorescence spectrum of PSI-LHCI (solid line) and PSI-LHCI-Lhcp (dotted line). (c) PSI light-harvesting capabilities in the A3 and A3L fractions. Light-induced P700 oxidation kinetics of PSI were measured in A3 and A3L fractions under 28 µmol photon m–2 s–1. The fraction of P700 oxidation was derived from Δ(A820-A870). Solid lines and shaded area represent averages of five (for A3) or ten (for A3L) technical replicates and SD, respectively. Blue lines represent fitting curves by mono-exponential functions. Data are representative of two biologically independent experiments. See another set of data in Figure 1—figure supplement 3. Figure 1—source data 1 Quantitative data for Figure 1c. https://cdn.elifesciences.org/articles/84488/elife-84488-fig1-data1-v2.zip Download elife-84488-fig1-data1-v2.zip Table 1 Pigment composition in the A3L fraction as revealed by UPLC analysis. Mean (±STD, n=3). PigmentMolar ratio (Chl a=100)Chl b36.2 (±0.1)β-Carotene13.4 (±0.5)Mdp3.8 (±0.1)Uriolide4.9 (±0.1)Prx11.8 (±0.1)Nex3.6 (±0.1)Vio3.3 (±0.1)Micromonal2.3 (±0.1)Dlt2.2 (±0.1) Fluorescence properties of A3 and A3L were characterized by fluorescence decay associated spectra at 77 K (FDAS, Figure 1—figure supplement 2a–c). The fluorescence lifetimes of A3 were mainly made up of <10 and 65 ps components (Figure 1—figure supplement 2b), which correspond to the total trapping time around P700, including the energy transfer between bulk Chl and P700 and the trapping at P700 (Mimuro et al., 2010). The positive peak in the fastest lifetime component observed in A3 was similar to that previously observed in the PSI core of cyanobacteria, which reflects the fast energy transfer process to P700 from the Chl near P700 and the subsequent fast charge separation. In A3L, however, the fastest lifetime component showed different shapes from that in A3 and it is also implying PSI-LHCI-Lhcp supercomplex in A3L. Because A3L has larger light-harvesting antennae, there are more Chls further away from P700 (e.g., those bound to Lhcp) than A3. Therefore, the long-range energy transfer process from the antennae to P700 becomes dominant in the observation, and the subsequent early trapping process could be masked. The slight increase in the lifetimes of A3L compared to those of A3 (<10–20 ps, 65–90 ps) likely reflects the presence of larger antennae in A3L. The light-induced oxidation kinetics of P700 indeed showed that the PSI antenna size in A3L was larger than that of A3 (Figure 1c, Figure 1—figure supplement 3). Notably, the 77 K fluorescence spectra of PSI-LHCI and PSI-LHCI-Lhcp exhibit a peak at 690 and 680 nm, respectively (Figure 1b), and there are no distinctive far-red peaks as reported previously (Swingley et al., 2010). The blue-shifted fluorescence was more prominent in PSI-LHCI-Lhcp fraction. These results and the EM analysis of the negatively stained particles (Figure 1—figure supplement 4, Table 2) suggest that A3 almost exclusively consists of PSI-LHCI supercomplex, while A3L was mainly composed of PSI-LHCI-Lhcp supercomplex. We thus proceeded to solve the cryo-EM structure of the large PSI-LHCI-Lhcp supercomplex in A3L in order to reveal its detailed architecture. Table 2 Two-dimensional classification of the photosystem I (PSI) particles in the A3 and A3L fractions. The RELION 2.1 package (Kimanius et al., 2016) was used for automated particle picking of particles and Two-dimensional (2D) classification into 50 classes as previously described (Watanabe et al., 2019). Classes of poor quality due to aggregation, contamination, micrograph edge, or extreme proximity were discarded. 2D classification was performed on the PSI particles and manually assigned to respective small and large PSI supercomplexes. FractionA3A3LSmall PSI (PSI-LHCI)2675 (100%)1030 (30%)Large PSI (PSI-LHCI-Lhcp)2437 (70%)Total PSI particles26753467 Supramolecular assembly of OtLhcp trimers (Trimers) with PSI-LHCI complex Through the single-particle cryo-EM method, the structure of OtPSI-LHCI-Lhcp supercomplex is solved at an overall resolution of 2.94 Å and the local regions of three Lhcp trimers (namely Trimers 1–3) are refined to 2.9–3.5 Å (Figure 2—figure supplement 1, Table 3). As shown in Figure 2, the OtPSI-LHCI-Lhcp supercomplex consists of a central PSI monomer encircled by six LHCIs and three Trimers. OtPSI contains two large core subunits (PsaA and PsaB), three subunits on the stromal surface (PsaC, PsaD, and PsaE) (Figure 2a–c), nine small membrane-embedded subunits (PsaF, PsaG, PsaH, PsaI, PsaJ, PsaK, PsaL, PsaM, PsaO), one subunit on the lumenal surface (PsaN) (Figure 2d–f). In the supercomplex, a total of 314 Chls (245 Chl a, 60 Chl b, 9 magnesium 2,4-divinylpheoporphyrin a5 monomethyl ester [Mdp], 104 carotenoids, 2 phylloquinones, and 3 Fe4S4 clusters have been located [Table 4]). The densities for representative Chl, carotenoid, and lipid molecules as well as two small subunits (PsaM and PsaN) are shown in Figure 2—figure supplement 2. To our best knowledge, the structure of OtPSI has the largest number of subunits among the structures of PSI known so far (including those from plants, green algae, diatom, red algae, and cyanobacteria, Figure 3). Figure 2 with 3 supplements see all Download asset Open asset Overall architecture of OtPSI-LHCI-Lhcp supercomplex. (a) Top view of the supercomplex from stromal side along membrane normal. (b) and (c) Two different side views of the supercomplex along the membrane plane. While the protein backbones are shown as cartoon models, the pigment and lipid molecules are presented as stick models. The iron-sulfur clusters are presented as sphere models. The bulk region of photosystem I (PSI) core is colored in wheat, while Lhca1-6, Lhcp1, and Lhcp2, PsaC, PsaD, PsaE, PsaF, and PsaN are highlighted in different colors. The remaining small subunits are in silver. (d–f) The small subunits at the interfaces between PSI core and LHCI/Lhcp complexes. The viewing angles are the same as (a–c), whereas the color codes are different. The interfacial small subunits are highlighted in various colors, while the PSI core and LHCI/Lhcp complexes are in silver. Figure 3 Download asset Open asset Comparison of O.tauri PSI-LHCI-Lhcp supercomplex with the photosystem I (PSI) supercomplexes from other organisms. (a) OtPSI-LHCI-Lhcp supercomplex. (b–k) Structures of PSI from different species or at different states. Blue, large PSI core subunits; magenta, small PSI core subunits; yellow, Lhca5-Lhca6/Lhca2-Lhca9/Lhca0-Lhca9/Lhc1-Lhc2 and the outer belt of LHCI; cyan, Lhca1-Lhca2-Lhca3-Lhca4 and the inner belt of antenna complexes around PSI from other species; green, Lhcp or LHCII trimers; gray, symmetry-related units in trimeric or tetrameric PSI. Table 3 Statistics of structural analysis of the OtPSI-LHCI-Lhcp supercomplex. OtPSI-LHCI-LhcpData collection and processingMagnification130,000Voltage (kV)300Electron exposure (e- Å–2)60Defocus range (μm)from –1.8 to –2.2Pixel size (Å)1.04Symmetry imposedC1Initial particle images (no.)5,288,217Final particle images (no.)80,366Map resolution (Å)2.94 FSC threshold0.143Map resolution range (Å)2.3–4.3RefinementInitial model used (PDB code)5ZJI, 7D0JModel resolution (Å)3.0 FSC threshold0.5Map sharpening B factor (Å2)–110.961Model composition Nonhydrogen atoms64,836 Protein residues5632 Ligands450B factor (Å2) Protein56.82 Ligand56.55R.m.s. deviations Bond lengths (Å)0.011 Bond angles (°)1.79Validation MolProbity score1.51 Clash score5.41 Poor rotamers (%)0Ramachandran plot Favored (%)96.6 Allowed (%)3.37 Outliers (%)0.04 Table 4 Summarization of the components in the final structural model of the OtPSI-LHCI-Lhcp supercomplex. SubunitNumber of amino acid residues tracedChlorophyllsCarotenoidsLipidsOthersPsaA74244 Chl a1 Cl06 BCR2 PG2 MGDG1 DGDG1 PQN1 Fe4S4PsaB73240 Chl a8 BCR1 PG1 DGDG1 PQNPsaC802 Fe4S4PsaD143PsaE62PsaF1653 Chl a1 BCRPsaG953 Chl a1 BCRPsaH963 Chl a1 SQDGPsaI351 BCRPsaJ411 Chl a1 BCR2 MGDGPsaK874 Chl a3 BCR1 PGPsaL1585 Chl a4 BCR1 PG1 MGDGPsaM31PsaN912 Chl aPsaO965 Chl a2 DLT2 MGDGTrimer 2201(Lhcp2)8 Chl a5 Chl b1 DVP2 PRX4 DLT1 NEX225(Lhcp1)8 Chl a5 Chl b1 DVP2 PRX4 DLT1 NEX201(Lhcp2)8 Chl a5 Chl b1 DVP2 PRX4 DLT1 NEXTrimer 1202(Lhcp2)8 Chl a5 Chl b1 DVP2 PRX4 DLT1 NEX201(Lhcp2)8 Chl a5 Chl b1 DVP2 PRX4 DLT201(Lhcp2)8 Chl a5 Chl b1 DVP1 BCR2 PRX3 DLTTrimer 3201(Lhcp2)8 Chl a5 Chl b1 DVP1 BCR2 PRX3 DLT200(Lhcp2)8 Chl a5 Chl b1 DVP2 PRX4 DLT1 NEX200(Lhcp2)8 Chl a5 Chl b1 DVP1 PRX4 DLTLhca11959 Chl a2 Chl b1 XAT1 PRXLhca220511 Chl a4 Chl b1 XAT1 BCR1 DLT1 PRX1 PG1 MGDGLhca322713 Chl a1 Chl b1 XAT3 BCR1 PRX3 PG1 MGDGLhca420511 Chl a4 Chl b1 XAT1 BCR1 PRXLhca51859 Chl a1 Chl b1 XAT1 PRX1 MGDGLhca621810 Chl a3 Chl b1 XAT1 PRX1 PG1 MGDG1 SQDGChain Y4 H2O Four Lhca (Lhca1-4) complexes are associated with the PSI core at the side of PsaG- F-J-K (Figure 2a and d), similar to those found in plant PSI-LHCI complexes (Mazor et al., 2017; Qin et al., 2015; Figure 3). On the other side formed by PsaG-I-M-H, an Lhca5-Lhca6 heterodimer is associated with the PSI core (Figure 2a and d). The location of Lhca5-Lhca6 heterodimer overlaps with that of Lhca9-Lhca2 heterodimer associated with C. reinhardtii PSI (Su et al., 2019; Suga et al., 2019) or Lhcr2-Lhcr1 heterodimer associated with Cyanidioschyzon merolae PSI (Pi et al., 2018), whereas the corresponding sites are vacant in plant PSI structures (Mazor et al., 2017; Pan et al., 2018; Qin et al., 2015; Yan et al., 2021). While the fourth transmembrane helix of CrLhca2 or Lhca5 from Dunaliella salina (also known as transmembrane helix F/TMF or TMH4), located at the dimerization interface of the Lhca2/Lhca9 or Lhca5-Lhca6 heterodimer, was previously proposed to replace the role of PsaM in mediating assembly of the LHCI heterodimer with PSI (Caspy et al., 2020; Suga et al., 2019), the fourth helix of OtLhca6 and PsaM coexist in O. tauri. As PsaM is also present in moss (Gorski et al., 2022) and cyanobacteria (Jordan et al., 2001), where LHCI heterodimer is absent at this position, the role and the origin of the fourth helix of the Lhca proteins might not be directly related to PsaM and need to be revisited. Previously, it was found that plant and C. reinhardtii PSI complexes contain some red-form Chls, absorbing photons at energy levels below that of the primary donor and mainly located in the LHCI complexes (Croce and van Amerongen, 2013). Unlike plant and C. reinhardtii PSI, OtPSI-LHCI-Lhcp supercomplex sample does not exhibit far-red peaks in the 77 K fluorescence spectra (Figure 1b). In the OtPSI-LHCI, the Chl a603-a609 pairs of Lhca1-4 (corresponding to the red-form Chls found in plant and C. reinhardtii Lhca1-4) are separated at larger distances, while those in Lhca5 and Lhca6 are similar to the ones in Lhca9 and Lhca2 from C. reinhardtii (Figure 4). Moreover, the axial ligands of Chl a603 in Lhca1-4 from O. tauri are all His residues instead of Asn (Figure 4b–e). Mutation of Asn to His for the ligand of Chl a603 in plant Lhca3 and Lhca4 led to absence of red spectral forms, while substitution of Asn for His in Lhca1 caused red shift of the fluorescence emission (Morosinotto et al., 2003). As His has longer side chain than Asn, the position of Chl a603 in OtLhca1-4 is located farther away from the protein backbone (in comparison with those from plant and C. reinhardtii LHCIs) so that the distance between Chl a603 and Chl a609 becomes larger and their excitonic coupling strength might be reduced as a result. The axial ligands of Chl a603 in Lhca5-Lhca6 dimer from O. tauri are both Asn residues same as those in the Lhca9-Lhca2 dimer on a similar location in C. reinhardtii (Figure 4b–e). Although Chl a603-a609 pairs of Lhca9 and Lhca2 were proposed to be responsible for the red spectral forms in C. reinhardtii (Mozzo et al., 2010), those in OtLhca5 and OtLhca6 with similar configuration do not cause the red spectral forms (Figure 4f and g), indicating that the presence of Asn residues at the axial ligand site is not sufficient to cause the spectral red form of Chl a603/a609. The distinct spectroscopic features of OtPSI-LHCI-Lhcp supercomplex might also be related to the local environments around chlorophyll molecules. For instance, Tyr69 and Tyr75 in OtLhca5 and OtLhca6 forms van der Waals contact with Chl a609Lhca5 and Chl a609Lhca6, respectively (Figure 4f and g). In comparison, these residues are replaced by tryptophan residues (Trp65) in C. reinhardtii Lhca9 and Lhca2. Previously, it was reported that tryptophan residues located nearby chlorophyll molecules may induce deformation of the tetrapyrrole macrocycle and cause red shift in the Qy absorption bands of chlorophylls (Bednarczyk et al., 2016). Mutation of a tryptophan residue (in van der Waals contacts with a bacteriochlorophyll) to a Tyr or Phe residue caused a blue shift of the Qy absorption peak in the core light-harvesting complex of Rhodobacter sphaeroids (Sturgis et al., 1997). Therefore, the occurrence of Tyr69 or Tyr75 residues (instead of Trp residues) around Chl a609Lhca5 or Chl a609Lhca6 may account (at least in part) for the lack of red spectral forms in the OtPSI-LHCI-Lhcp supercomplex. Figure 4 Download asset Open asset Comparison of Chl a603-a609 dimers in Lhca complexes among O. tauri, C. reinhardtii, and P. sativum. (a) Overall arrangement of the Chl a603-a609 dimers in six Lhca complexes of O. tauri, C. reinhardtii, and P. sativum. The chlorophylls in the photosystem I (PSI) supercomplexes are presented in surface models and those of Lhca complexes are superposed with stick models. Color code: pink, O. tauri; golden, P. sativum; light blue, C. reinhardtii. The dark labels (1–6) indicate the Lhca1-Lhca6 subunits and the dashed ovals label the locations of the a603-a609 dimer in each LHCI. (b–g) Comparison of the Chl a603-a609 dimers and their local environments in six different Lhca subunits from the three different species. Note that the axial ligands of Chl a603 in Lhca1-4 from O. tauri are all His, while the Asn in the 603 site of plant Lhca3 and Lhca4 are crucial for the formation of the red-most form chlorophyll. The Lhca5/Lhca9 and Lhca6/Lhca2 are only present in O. tauri and C. reinhardtii but absent in P. sativum. The Chl a603-a609 dimers are indicated in the black dashed ovals and the key amino acid residues around the two chlorophylls are shown as stick models. The number labeled nearby the black dashed lines indicate the Mg-Mg distances between Chl a603-a609 dimer in Lhca complexes from O. tauri, C. reinhardtii, and P. sativum. In (f) and (g), the red dashed ovals indicate the Tyr/Trp residues around the Chl a609 molecules from OtLhca5/CrLhca9 and OtLhca6/CrLhca2. Besides the six Lhca proteins, three Lhcp Trimers (Trimers 1–3) bind to the PSI-LHCI complex on the third side along the surfaces of Lhca6, PsaH, PsaL, PsaO, and PsaK subunits (Figure 2a and d). As a result, the PSI core is enclosed by an irregular annular belt formed by the LHCI, Lhcp complexes, PsaG and PsaK (Figure 2a and d). This side of the PSI core was partly filled by one LHC trimer or two LHC trimers in higher plants or green algae when they are under state 2 conditions (Huang et al., 2021; Pan et al., 2018; Pan et al., 2021). Among the three trimers associated with OtPSI, Trimers 1 and 3 are both (Lhcp2)3 homotrimers, whereas Trimer 2 is a Lhcp1(Lhcp2)2 heterotrimer. The detailed cryo-EM map features for identification of Lhcp1 and Lhcp2 are shown in Figure 2—figure supplement 3. Trimers 1 and 3 bind to PSI on the PsaO and PsaK sides, respectively, while Trimer 2 assembles with PSI on the PsaL-PsaH side through Lhcp1 subunit and interacts with Lhca6 through an Lhcp2 subunit. As Trimer 1 is sandwiched between Trimers 2 and 3, it forms close contacts with both Trimers 2 and 3, and is related with them through pseudo-C2 symmetry axes at their interfaces. In C. reinhardtii, two LHCII trimers associate with PSI in state 2 (Huang et al., 2021; Pan et al., 2021), whereas one LHCII trimer is located at the peripheral region of Zea mays PSI (ZmPSI) in state 2 (Pan et al., 2018; Figure 3). While the binding sites of Trimers 1 and 2 partially overlaps with those of LHCII-1 and LHCII-2 trimers from CrPSI-LHCI-LHCII supercomplexes respectively, they do not superpose well with each other (Figure 5). When the PSI core regions are aligned, O. tauri Trimer 1 appears to be rotated by 57 degrees and shifted by 16.1 Å in relation to the position of C. reinhardtii LHCII-1. Trimer 2 is rotated by 52 degrees in relation to the position of C. reinhardtii LHCII-2. The LHCII trimer associated with ZmPSI binds to a position between Lhcp Trimers 1 and 3. Figure 5 Download asset Open asset Comparing the binding sites of Trimers with those of LHCII trimers bound to C. reinhardtii and plant photosystem I (PSI). (a and b) The structure of OtPSI-LHCI-Lhcp supercomplex is superposed with the PSI-LHCI-LHCII supercomplex from C. reinhardtii (PDB code: 7DZ7, a) or Z. mays (PDB code: 5ZJI, b). The three structures are superposed on the common PsaA subunits. The dash triangular rings outline the approximate boundaries of Trimers or LHCII trimers. Color code: green, OtPSI-LHCI-Lhcp; blue, CrPSI-LHCI-LHCII; magenta, ZmPSI-LHCI-LHCII. The double-headed arrows indicate the translational or rotational relationships between Lhcp trimers in O. tauri and the corresponding LHCII trimers in C. reinhardtii and in Z. mays. The number labeled nearby the arrows indicate the translation distances or rotation angles. Trimer 1 is located nearby PsaO and the closest interfacial distances between them are 7.0 Å or larger (Figure 6a). Although Trimer 1 does not form direct interactions with PsaO, we cannot rule out the presence of unresolved lipid molecules that may mediate the interactions. On the other hand, Trimers 2 and 3 form close and direct interactions with PsaL-PsaH and PsaK, respectively (Figure 6b and c). Trimer 2 binds to PSI on three different sites (Figure 6d–f). At site 1, Lhcp1Trimer2 has its elongated N-terminal region (NTR) partially inserted into a surface pocket formed by PsaL and PsaH subunits (Figure 6b). The NTR of Lhcp1 contains an RRpT (pT, phosphorylated Thr residue) motif identical to those found in Z. mays pLhcb2 (Pan et al., 2018) and C. reinhardtii pLhcbM1 (Pan et al., 2021). The RRpT motif of Lhcp1 interacts with nearby amino acid residues through salt bridges and hydrogen bonds (Figure 6d), in a similar way as those of Z. mays pLhcb2 and C. reinhardtii pLhcbM1. Arg29 in Lhcp1 might be acetylated on its main-chain amine group as there is an extra density linked to it, and the van der Waals interactions between the putative acetyl group and nearby amino acid residues from PsaH serve to stabilize the association between Lhcp1 and the PSI core (Figure 6d). Consistently, spinach Lhcb proteins are also acetylated at their amino-terminal arginine and phosphorylated on threonine/serine in the third position (Michel et al., 1991). Sites 2 and 3 are located in the membrane-embedded regions on the stromal and lumenal sides, respectively (Figure 6b). On these two sites, Lhcp1 forms hydrogen bonds and van der Waals interactions with nearby amino acid residues and pigment molecules from PsaH and PsaL subunits (Figure 6e and f). Meanwhile, Trimer 3 associates with PsaK mainly through van der Waals interactions on the stromal and lumenal sides (Figure 6g). Figure 6 Download asset Open asset Interfaces between Trimers and photosystem I (PSI) subunits. (a) The interface between Trimer 1 and PsaO. (b) The interface between Trimer 2 and PsaL-PsaH. (c) The interface between Trimer 3 and PsaK. (d–f) The detailed interactions between Lhcp1 of Trimer 2 and PsaL/PsaH at sites 1–3 shown in b. (g) The detailed interactions between Lhcp2 of Trimer 3 and PsaK at site 4 shown in c. The numbers labeled nearby the dash lines are distances (Å) between two adjacent groups. Characteristic features of Lhcp1 and Lhcp2 monomers As an ancient member of the green lineage, O. tauri belongs to Mamiellales of Prasinophyceae at the basal position of the green lineage. While OtLhcp1 and OtLhcp2 are evolutionarily related to plant Lhcbs and CrLhcbMs (Six et al., 2005), they only share low sequence identity, e.g., 32–37% sequence identity with ZmLhcb2 or CrLhcbM1. The apoproteins of OtLhcp1 and OtLhcp2 adopt a classical fold of Lhc family with three transmembrane helices (A, B, and C) and three short amphipathic helices on the lumenal side (D, E, and F) (Figure 7a and b and Figure 7—figure supplement 1). While the structure of OtLhcp1 highly resembles that of OtLhcp2, it differs from those of ZmLhcb2 and CrLhcbM1 in the NTR, EC loop, AC loop, and CTR (Figure 7—figure supplement 2a–c). Besides, helices B, A, and C of OtLhcp1/2 are slightly shorter than the corresponding ones in ZmLhcb2 or CrLhcbM1 (Figure 7—figure supplement 2b, c). Figure 7 with 2 supplements see all Download asset Open asset The structures and pigment compositions of OtLhcp1 and OtLhcp2. (a and b) Side views of OtLhcp1 (a) and OtLhcp2 (b) structures. The backbones of OtLhcp1/OtLhcp2 apoproteins are showed as cartoon models, while the pigments are presented as stick models. The phytyl chains of Chl molecules are omitted for clarity. The phosphorylated Thr residue and the acetylated Arg residue at the N-terminal region of Lhcp1 is highlighted as sphere models. A, B, and C indicate the three transmembrane helices in OtLhcp1/OtLhcp2 apoproteins, whereas D and E are the two amphipathic helices at the lumenal surface. Color codes: gray, Lhcp1 and Lhcp2 apoproteins; green, Chl a; cyan, Chl b; magenta, dihydrolutein/DLT; purple, prasinoxanthin/PRX; orange, neoxanthin/NEX. (c and d) The arrangement of pigment molecules in OtLhcp1 (c) and OtLhcp2 (d). For clarity, the apoproteins are not shown. In the upper row, only pigment molecules within the layer close to stromal surface are shown, while the lower row shows the pigment molecules within the layer close to the lumenal surface. In terms of pigment composition, OtLhcp1 and OtLhcp2 each contain 14 Chl molecules (8 Chl a, 5 Chl b, and 1 Mdp) and 7 carotenoid molecules (4 Dlt, 2 Prx, and 1 Nex) (Figure 7a and b). The Chl b/a ratio of the structural model is 0.625, close to the previously reported value of 0.736 for the Lhcp2 preparation (Swingley et al., 2010). While the number of Prx molecules found in the structure matches the previous prediction, four (instead of one) Dlt molecules are more than expected (Swingley et al., 2010). As O. tauri OTH95 species thrives in lagoons and shallow area of the ocean frequently challenged by high light with an intensity up to 200