Cryo-EM Structure of a Tetrameric Photosystem I from Chroococcidiopsis TS-821, a Thermophilic, Non-heterocyst-forming Cyanobacteria


 Photosystem I (PSI) is one of two the photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, which is in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutional significance. Here we report the ~3.9 Å resolution cryo-EM structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsis sp. TS-821. The structure resolves all 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.


47
Oxygenic photosynthesis is a unique energy conversion process performed by plants, algae, and 48 cyanobacteria (1, 2) where photons from the sunlight are converted into chemically fixed energy 49 by synthesizing carbohydrates, generating oxygen as a side-product of water splitting. Oxygen 50 production and carbon dioxide fixation into organic matter performed by photosynthetic organisms 51 determines the composition of Earth's atmosphere and provides all life forms with essential food 52 and fuel (3,4). Oxygenic photosynthesis of cyanobacteria, algae, and plants is catalyzed by four 53 defined membrane complexes: PSI, photosystem II (PSII), cytochrome b6/f complex, and CF1- 54 ATPase, which are the major components of the electron transport chain (ETC). Both PSI and PSII 55 and unicellular cyanobacteria, including the most primitive known cyanobacterium, Gloeobacter 79 violaceus PCC 7421 (14)(15)(16)(17). Eventually, the trimeric PSI structure was resolved at 2.5 Å by X-80 ray crystallography from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 81 (T.e. BP-1) (PDB ID: 1JB0), confirming the conclusions about the trimeric structure of PSI (9).

82
As a result of these early seminal reports, PSI was initially believed to assemble into stable trimeric 83 structures in cyanobacteria, as opposed to the monomeric form observed in all plants and algae. 84 Since then, further evidence for trimeric PSI has been observed in the 2.5 Å crystal structure of 85 the mesophilic cyanobacteria Synechocystis sp. PCC 6803 (Syn PCC 6803) (PDB ID: 5OY0) (18) 86 and recently in atomic force microscopy analysis of multiple ecotypes of Prochlorococcus (19), 87 suggesting a prevalent occurrence of trimeric PSI in cyanobacteria.

89
In cyanobacteria, a PSI protomer comprises twelve different subunits, and in many species, the 90 total mass of a trimer is ~1 MDa (9,10,20). However, this initial belief of PSI trimer being the 91 sole oligomeric state in cyanobacteria has recently been challenged by observation of the tetram-92 eric form of PSI in two cyanobacteria Anabaena sp. PCC 7120 (21,22) and Chroococcidiopsis sp. 93 TS-821 (TS-821) (23). However, the PSI tetramer was not considered as a major oligomeric state 94 in cyanobacteria. The physiological and evolutionary significance of this tetrameric state has yet 95 to be elucidated, nor the mechanism driving this oligomerization state and sustaining its stability 96 is known. The recent cryo-EM structure of PSI tetramer from TS-821 revealed that tetrameric PSI 97 is actually a dimer-of-dimers with two different interaction interfaces between monomers (24). 98 This structure suggests that subtle changes in the placement of the central PsaL subunit yield 99 changes in helical bundling that have been implicated to be critical in the formation of PSI trimers. To date, no crystal structure of the tetrameric PSI is available. Although two cryo-EM structures 102 of the tetrameric form of PSI has been reported in a heterocyst-forming cyanobacterium, Anabaena 103 (21,22), our lab has observed that a tetrameric PSI organization is very widespread being found    (Fig. 1E). TEM imaging showed a thin section of two cells revealing thick outer 140 sheath composed of a fibrous outer cell wall layer or F-layer, as initially observed in the Pleu-141 rocapsales (28).

142
Observation of cells under bright field microscopy revealed that TS-821 cells exist primarily as 143 one or two cells or in larger aggregates (Fig. 1C). Cells were also stained with DAPI, a DNA 144 specific fluorescent dye, in order to observe the location of DNA within cells during division (Fig. 145 1D). The cell undergoing binary fission exhibits fluorescence throughout the entire interior portion 146 of the cell, suggesting that DNA is distributed throughout the cell (Fig. 1D). However, distinct 147 regions of fluorescence were seen within adjacent cells dividing via multiple fissions. These dis-148 tinct globular regions of DNA are most likely small daughter cells that are the result of multiple 149 fissions. All of this imaging supports the original morphological classification of TS-821 as a 150 member of the order Pleurocapsales (28).

151
To analyze the phylogeny of strain TS-821, a phylogenetic tree was generated as described in 152 materials and methods. It is also important to note that genera representing all major orders within 153 the phylum Cyanophyta are included within this phylogenetic analysis. Fig. 1F  Although our earlier cryo-EM low-resolution structure suggested TS-821 tetramer was a dimer-165 of-dimers, this resolution prevented us from investigating the structural basis of this unique sym-166 metry (24). To have a better insight into the organization of tetrameric PSI from TS-821 we con-167 ducted the single-particle cryogenic electron microscopy analysis (Fig. S1A). 2D particle classifi-168 cation of 325,648 particles yielded 16 different 2D classifications (Fig. S1B) which upon relion 169 3D classification/refinement yielded 63,131 particles (Fig. S1C). From this classification, we ob-170 tained ~3.9 Å resolution structure ( Fig. S1D-H). 171 The local resolution of the final map varies from 3.5 Å to 5.5 Å (Fig. S1E) with high resolution 172 within the transmembrane core of each monomer composed of multiple PsaA and PsaB helices, 173 suggesting increased protein stability and less conformational flexibility. In addition, the interfa-174 cial subunits PsaL, PsaI and PsaM at one of the dimeric interfaces contain the best resolution dis-175 tribution within the map, possibly reflecting higher stability.

176
To reconstruct a tetramer of PSI, the single protomer of cyanobacterial PSI (PDB ID: 1JB0) was 177 manually placed in the cryo-EM map using Chimera (29).This rough placement was followed by 178 the rigid-body refinement of each subunit in Phenix (30). For all subunits we mapped density for 179 all known PSI subunits except for PsaX, which had a very fragmented and weak density. This 180 could indicate that this subunit may have been partially lost during the sample preparation. Thus,

181
PsaX was not included in further modeling. The sequence was manually adjusted during the mod- As we have previously shown (24), the tetramer is organized as a dimer-of-dimers with two dif-190 ferent interfaces: A-B and B-A' (Fig. 2A, S3A-B). Therefore, the structure is pseudotetrameric 191 and it has C2 and not C4 symmetry. The obtained cryo-EM structure of the tetrameric PSI of TS-192 821 resolves all 44 subunits: four each of PsaA,PsaB,PsaC,PsaD,PsaE,PsaF,PsaI,PsaJ,PsaK,193 PsaL, and PsaM (Fig 2B-C). The inner cavity is surrounded by four PsaL, two PsaM, and two PsaI 194 subunits. The relative positions of the subunits are identical in the individual monomers. However, 195 the tetramer has a dual symmetry where the positioning of the monomers A and B are identical to 196 that of monomers A' and B' supporting the hypothesis that tetrameric PSI is a dimer-of-dimers.

197
The PsaL subunits are closely associated with each other between monomers A-B and A'-B' (Fig. 198  (PDB ID-1JB0), we have been able to putatively place both phylloquinones and carotenoids in 210 our model. Each monomer has two phylloquinones and 16 β-carotene molecules that were manu-211 ally placed (Fig. 2D-E). However, the identity of these carotenoids could not be matched to the 212 prior chemically identified carotenoids (25). There are a total of 362 Chl a molecules in the te-213 tramer, 92 in monomers A and A' and 91 in monomers B and B'. The special pair of Chl a is 214 located at the center of the PsaA-PsaB interface, towards the lumenal side ( Fig. 2D-E). 215 Closely placed Chl a molecules with Mg 2+ -Mg 2+ distance < 10 Å are found primarily within indi-216 vidual monomers but are not found in any of the four interfaces between monomers (Fig. S2A). A 217 pair of Chl a molecules has one Mg 2+ -Mg 2+ distance of less than 15 Å at both the A-B and A'B' 218 interfaces (Fig. S2B). There are no Chl a molecules with Mg 2+ -Mg 2+ distances < 15 Å across the 219 B-A' and B'-A interfaces (Fig. S2B). There are many Chls within < 20 Å in all the interface (Fig.  Chl a's oriented parallel to one another near the monomer interface (Fig. S2F). 224 Central cavity 225 The central cavity of the tetramer contains unresolved densities which may correspond to lipids, 226 detergent molecules, or pigment/carotenoid molecules. A model cyanobacterial membrane con-227 taining 47% monogalactosyldiacylglycerol (MGDG), 23% digalactosyldiacylglycerol (DGDG),

231
The central cavity is about 70 Å x 50 Å and has an approximate volume of 65,000 Å 3 (Fig. 3C

248
The A-B interface contains more proximal Chl (11) than the B-A' interface (6). However, the B-

249
A interface includes more transmembrane domains (14) than the A-B interface (7). This composi-250 tional difference is reflected in the increased buried surface area of A-B (2,418 Å 2 ) whereas the B-

251
A' only has 1561 Å 2 (Fig. S3D). In addition, we compared interfaces with missing Chl a mole-252 cules to assess the impact of the Chl a molecules. Overall, Chl a molecules contribute to the solv-253 ation energies of both interfaces and the regulation of electrostatic energies. As expected, they also 254 contribute to the larger surface area for the A-B and A'-B' interfaces, indicating that they are 255 integral for their formation and stability.

283
Early work has shown that PsaL is key to the assembly and stability of the trimeric structure of 284 PSI (32). Since the A-B interface in the tetrameric PSI resembles the trimeric PSI interface at the 285 core, we investigated the interaction interface of PsaLs in PSIs of both T.e. BP-1 and TS-821 and 286 other tetramers (PDB IDs 6JEO and 6K61) (Fig. 5). The three PsaL subunits form a central helical 287 bundle in T. e. BP-1 PSI trimer (Fig. 5A). This central core contains a PsaL subunit from each PSI 288 monomer and has virtually no cavity. In the trimer, the enlarged view highlights the residues that 289 stabilize this PsaL bundle. These residues span the entire transmembrane region with mostly non- anobacteria. This phylogenetic approach is both broader and based on a different evolutionary trait 304 from either the initial tree based on only 16S rRNA (Fig. 1A) or the previous tree utilizing 29 305 universally conserved genes (25). Moreover, this phylogenetic tree is focused on changes associ-306 ated with the PsaL (Fig. 6A). 307 Our analysis of a non-redundant set of PsaL sequences yielded four putative monophyletic clusters 308 (Fig. 6A). Each cluster does contain multiple species whose PSI oligomeric state/type is experi-309 mentally known (trimeric, tetrameric, or far-red), giving us further confidence in this phylogenetic 310 approach to identify PSI oligomerization states. In some cases, organisms contained multiple PsaL 311 proteins that fit into two or three groups. Classification of each group was based on existing struc-312 tural data from representative members: using Leptolyngbya sp. strain JSC-1 (33) as an anchor for 313 the far-red cluster, T. e. BP-1 as trimer cluster (9)  The sequence LogoPlots of the trimer and far-red sequences are most alike based on conserved 321 regions (CR) across CR-I and CR-II in the linker region (Fig. 6B). Similarly, the far-red and tri-322 mers show signs of common ancestry based on the c-terminal regions, specially CR-III and CR-323 IV. Interestingly, the tetramers were most similar to the Prochlorococcus/Synechococcus group 324 based on their linker region with conserved TV/T/APNPP motif found in CR-V region (Fig. 6C). 325 Unlike the trimers and far-red groups, there were no conserved regions between tetramers and the In this study, the structure of tetrameric PSI from TS-821, a non-heterocyst forming cyanobacteria, 346 was solved by cryo-EM with a resolution of 3.9 Å resolution and has an organization of a dimer- of PsaL in the AB interface has multiple bulky (mostly non-polar and aromatic) residues while the 363 stromal face has polar residues. This is similar to that of the tetrameric PSI of heterocyst-forming 364 cyanobacteria, where it was suggested that specific amino acids with large side chains may prevent 365 formation of the trimer due to the steric hindrance provided by these bulky groups (22).

366
Our work adds a new tetrameric PSI cryo-EM structure for a cyanobacteria outside of the two 367 reports in Anabaena sp. PCC 7120 (21,22). This work coupled with our prior BN-PAGE and 368 bioinformatic characterization (23, 24) suggest that most if not all members of the HCR group of 369 cyanobacteria have a tetrameric form of PSI. However, the evolutionary role of this change in PSI 370 structure is still elusive. Previous work has shown that in three different cyanobacteria, exposure 371 to high light can induce the formation of tetrameric PSI and was also shown to induce the accu-372 mulation of more novel carotenoids in the thylakoid membranes (25). This might suggest that one 373 role of the tetramer is to allow accumulation of photoprotective carotenoids with PSI when 374 exposed to high light environments. How these carotenoids are associated with PSI is unknown 375 but their release upon dissociation into two dimers suggests that on possibility is an association 376 within the central ~ 65,000 Å 3 central cavity.

377
The subunit PsaL was found to be important for formation of the trimeric form of PSI (32). Our 378 bioinformatic work has identified small conserved regions in PsaL that correlate with this tetram-379 eric symmetry by promoting PsaL dimerization versus a trimerization in the PSI timers. According  The TS-821 thylakoid membrane containing 1 mg/mL Chl a were solubilized in 1% β-DDM (Gly-472 con Biochemicals, Luckenwalde, Germany). The solubilized membrane solution was loaded on a 473 10-30% sucrose gradient containing 0.01% β-DDM in the wash buffer. Two-step ultracentrifuga-474 tion was used to purify PSI tetramer as described previously (48)   The fixation protocol is the same as described above until the dehydration step. Samples were then 500 washed in water 3X before dehydration in a graded ethanol series, and then finally dehydrated  Orange Capped Lysis Matrix A Tubes, which are specific for DNA isolation. A large cell pellet 511 harvested from a dense liquid cell culture was resuspended in 1 mL 1x TE buffer (10 mM, Tris-512 HCl, pH 7.5, 1 mM EDTA) and homogenized at 4 m/s for 20 s. Lysate appeared blue due to the 513 release of phycobilin proteins into solution. This tube was centrifuged for 1 min at 10,000 g, and 514 the supernatant was immediately transferred to another1.5 mL, microfuge tube. All additional steps 515 were done, as previously described (50). Due to a significant amount of RNA contamination, 3 μL 516 of RNase (10 mg/mL) was added to each tube and allowed to incubate at room temperature for 10 517 min. Amplification of the 16S rRNA was accomplished using these primers: forward (5'-AGAG-518 TTTGATCCTGGCTCAG-3') and reverse (5'-AAGGAGGTGATCCARCCGCA-3'). The 50 μL 519 reaction mixture consisted of 10 μL of 5X GoTaq Reaction Buffer, 1 μL dNTP mixture (10 mM 520 each), 1 μL of each primer (10 pM/μL), 0.25μL GoTaq Polymerase, 50 ng of template DNA and 521 an appropriate amount of nuclease-free water to achieve 50 μL total reaction. The PCR conditions 522 were as follows: 95° for 3 min, 30 cycles of 95°C for 1 min, 60°C for 1 min, 72°C for 1.5 min, and 523 finally 72°C for 10 min subsequently followed by a 4°C hold. The resultant PCR product was 524 cleaned up using QIAquick® PCR clean-up system and immediately ligated into the TOPO   phages. Based on the whole genome sequences, a subset of these 1,639 genomes was generated by 618 building a Mash tree (82) to reduce redundancy. We selected a cutoff value of 0.1 based on the 619 first plateau in the plot (Fig. S1), which resulted in 295 clusters. In some of these clusters, we 620 observe sets of highly related or even identical organisms (data not shown), so we selected a unique 621 yet representative member from each of the 295 clusters, using a random number generation from 622 the Python package NumPy (83). This allowed us to randomly choose a single genome from each 623 cluster yet also significantly reduced redundancy from 1,639 to only 295 distinct non-redundant 624 organisms.          Interaction interface of PsaL subunits in trimeric (T. e. BP1) and tetrameric (6JEO and 6K61). A) highlights the polar and non-polar residues that participate in the interaction interface at the interface of the three PsaLs of their respective monomers in T. e. BP-1. B) highlights the residues participating in the interaction interface between the two PsaLs of the A-B or A'-B' interface, RMSD-0.708 (Nostoc and Anabaena aligned) and RMSD-5.732 (TS-821 and Nostoc aligned). C) Shows the PsaL from all three tetramers (TS821, Nostoc and Anabaena) A-B interface (stromal and membrane view). It also shows the alignment of all the Chl a molecules in the three tetramers.

Figure 6
Phylogenetic Analysis of PsaL and motif-analysis. A) This represents a maximum-likelihood tree built using FastTree 2 on the multiple alignment of the 108 PsaL proteins using MUSCLE. The color shaded regions delineate separate groups that are known to include different forms of PSI including trimeric forms of PSI, far-red light forms of PSI, tetrameric forms of PSI, and a fourth group of marine cyanobacteria including members of Prochlorococcus and Synechococcus. Some species contain multiple copies of PsaL in their genome and these are denoted by colored circles (by species) and numbers (by number of PsaL copies). B) Logo plot of the loop region between the predicted TMD #2 and #3. The ending and beginning of the TMD are shown by colored boxes below the Logo sequence. The bit score scale was set to 6 bits to allow the error bars to be visible. The conserved motifs were shaded and named CR I-V. Within each CR the most conserved amino acids were further indicated by an asterisk above the single letter code. C) Similar to panel B, we display the Logo plots of the PsaL C-terminus of the four different groups also indicating the conserved regions (CR III-IV). D) Comparison of the CRs in TS-821, T. e. BP-1 and Syn PCC 6803 PSIs.